Oculospanin as a tumor specific antigen and methods and compositions utilizing same

ABSTRACT

The present invention relates to a method of detecting cancer by use of an oncogene, a method of screening for an active compound useful to treat and/or prevent cancer, and a pharmaceutical composition for treatment and/or prevention of cancer. More specifically, the present invention provides a method of detecting cancer based on the expression of the human oculospanin gene as a marker and a pharmaceutical composition containing an antibody capable of specifically

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 11/223,812, filed Sep. 9, 2005, which is acontinuation of International Application No. PCT/JP2004/003048, filedMar. 9, 2004, which claims the benefit under 35 U.S.C. 365(c) ofJapanese Application No. 2003-063648, filed Mar. 10, 2003; and of U.S.Application No. 10/548,688, filed with the U.S. Patent Office on Sep. 9,2005, under 35 U.S.C. 371 from International Application No.PCT/JP2004/003048, filed Mar. 9, 2004, which claims the benefit under 35U.S.C. 365(c) of Japanese Application No. 2003-063648. The entiredisclosure of the above-listed prior applications is considered to bepart of the disclosure of the instant application and is herebyincorporated by reference therein.

FIELD OF THE INVENTION

The present invention relates to compounds such as an antibodies usefulin cancer treatment, a pharmaceutical composition for treating cancercharacterized in that it contains the antibody as an active ingredient,a method of detecting cancer and a cancer detection kit.

BACKGROUND OF THE INVENTION

Tumor cells are known to express antigenic proteins which are intrinsicto the particular type of tumor cells (hereinafter sometimes referred toas a “tumor-associated antigens”). Attempts have been made to developnew therapies for treating tumors by targeting tumor-associatedantigens. Monoclonal antibodies that elicit an antigen-antibody responsespecific to such tumor-associated antigens are known to induce varioustypes of in vivo immune responses (antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), etc.)to attack cancer cells, thereby inducing cell death. Monoclonalantibodies useful for tumor treatment have been developed.

However, the range of monoclonal antibodies useful for tumor treatmentis limited. The monoclonal antibodies presently available are capable oftreating only a few types of tumors including metastatic breast cancer,acute leukemic myelosis, intractable chronic lymphoma, non-Hodgkin'slymphoma, and multiple myeloma. Development of monoclonal antibodiesapplicable to treatment of other tumors is desirable.

To obtain a monoclonal antibody useful for tumor treatment, it isnecessary to identify a protein specifically expressed in a tumor celland obtain a monoclonal antibody against this protein antigen.

Human oculospanin protein was obtained as an Expressed Sequence Tag(EST) clone derived from a gene expressed on the retinal pigmentepithelium and the ocular choroidal membrane (Molecular Vision (2002) 8,25-220). The human oculospanin gene has an open reading frame of 1068bp. Human oculospanin consists of 355 amino acids and is estimated tohave a molecular weight of 36.4 kDa based on the DNA sequence. However,the relationship between human oculospanin and tumors is still unknown.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the present invention provides antibodieswhich specifically bind to a human oculospanin protein selected from atleast one member of the group consisting of SEQ ID NO:2 and SEQ ID NO:4,and b) has cytotoxic activity against a cell expressing the oculospaninprotein. In some aspects, the antibodies include chimeric antibodies(including humanized antibodies), human antibodies, monoclonalantibodies, and derivatives thereof), which can be IgG antibodies andpreferably IgG1 antibodies.

In a further aspect, the invention provides pharmaceutical compositionscomprising the antibodies of the invention and a pharmaceuticallyacceptable carrier.

In an additional aspect, the invention provides methods of screening forbinding to human oculospanin comprising contacting a human oculospaninprotein with a library of candidate agents and determining the presenceor absence of binding of at least one candidate agent and theoculospanin protein. In some aspects, either the protein or thecandidate agent can be immobilized on a solid support, includingmicrospheres. Either component can be labeled, for example withfluorophores.

In a further aspect, the invention provides methods of screening forcytotoxicity induction in a population of cells expressing a humanoculospanin protein comprising contacting the cells with a candidateagent to form a mixture and assaying for cytotoxicity. In some aspects,a library of candidate agents are tested, and in certain aspects, thecandidate agents are antibodies. The method optionally includes addingeffector cells to the mixture.

In an additional aspect, the invention provides methods of inducingcytotoxicity in a cell expressing human oculospanin comprising adding anagent that inhibits oculospanin activity such that cytotoxicity isinduced.

In a further aspect, the invention provides methods of detecting cancercomprising measuring the amount of nucleic acid encoding oculospaninfrom a human test sample, measuring the amount of nucleic acid encodingoculospanin from a human healthy sample, and comparing the difference inthe amounts to determine the presence of cancer in the test sample.

In yet another aspect, the invention provides methods of detectingcancer comprising measuring the amount of oculospanin protein from ahuman test sample, measuring the amount of oculospanin protein from ahuman healthy sample and comparing the difference in the amounts todetermine the presence of cancer in the test sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, the upper figure, is a graph showing the expression level of thehuman oculospanin gene in various types of cells; and the lower figureis a graph showing the expression level of the human oculospanin gene ina healthy person's skin samples and in melanoma samples;

FIG. 2, the upper figure, is a graph showing the expression level of thehuman oculospanin gene in a healthy person's skin samples and inmelanoma samples derived from skin tissue; and the lower figure is agraph showing the expression level of the human oculospanin gene in ahealthy person's skin samples and in melanoma samples derived from lymphnode tissue;

FIG. 3 is a graph showing the expression level of the human oculospaningene in samples derived from a healthy person's lymph node and inmelanoma samples derived from lymph node tissue;

FIG. 4 shows expression of human oculospanin gene products in NIH3T3cells; and

FIG. 5 is a graph showing antibody-dependent cytotoxic activity of ananti-human oculospanin antibody in a human oculospanin-expressing cell.

DETAILED DESCRIPTION OF THE INVENTION

1. General Overview of the Invention

The present invention is directed to the finding that the proteinoculospanin is overexpressed in certain cancer cells, particularlymelanoma, and that compounds that bind to oculospanin, such as antigenbinding proteins including antibodies, induces cytotoxicity in cellsexpressing oculospanin. Thus the present invention providescompositions, including antibodies, which induce cytotoxicity inoculospanin-expressing cells, and methods of diagnosing cancer as wellas methods of inducing cytotoxicity.

The identification of this correlation further allows a number ofmethods utilizing the oculospanin protein, including methods ofscreening for candidate agents that bind to and/or modulate the activityof oculospanin, including screening assays for candidate agents such asantibodies and/or other compounds for cytotoxicity of cells that expressoculospanin. As is more fully described below, these assays can take ona number of formats, including the use of substantially pure oculospaninproteins (including fragments and derivatives thereof) in homogeneousand heterogeneous assays, biochemical assays, and cellular assays thatutilize cells expressing human oculospanin proteins.

In addition, the invention provides for methods of inducing cytotoxicitythrough the use of agents that bind to oculospanin, including, but notlimited to, antigen binding proteins such as antibodies.

Accordingly, in one embodiment, the present invention provides antigenbinding proteins that bind to human oculospanin.

2. Oculospanin Proteins

By “human oculospanin protein” herein is meant the protein sequencedepicted in SEQ ID NO:2 and/or SEQ ID NO:4, and allelic variationsthereof. In some embodiments, for example when the oculospanin is usedin screening, fragments or derivatives of oculospanin protein (andnucleic acids, as outlined below) can be used. Thus, variants of humanoculospanin can be used in some embodiments, both for screening and forthe generation of antibodies, as well as other methods contemplatedherein. As is more fully outlined below, it may be desirable to usefusion oculospanin proteins that contain labels such as epitope tags forattachment to surfaces for screening, or labels such as autofluorescentproteins (e.g. green fluorescent proteins). In some cases, for examplein screening assays, it may be useful to use non-human oculospaninproteins, such as rodent or other non-human mammalian proteins.

2A. Oculospanin Variants

In some embodiments, depending on the use of the oculospanin protein,variants can be used. These variants fall into one or more of threeclasses: substitutional, insertional or deletional variants. Thesevariants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the oculospanin protein, using cassetteor PCR mutagenesis or other techniques well known in the art, to produceDNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture as outlined above. However, variant oculospaninprotein fragments having up to about 100-150 residues may be prepared byin vitro synthesis using established techniques. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the oculospanin protein amino acid sequence.The variants typically exhibit the same qualitative biological activityas the naturally occurring analogue, although variants can also beselected which have modified characteristics as will be more fullyoutlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed oculospanin variants screenedfor the optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays ofoculospanin protein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the oculospanin protein aredesired, substitutions are generally made in accordance with thefollowing chart: CHART I Original Residue Exemplary Substitutions AlaSer Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro HisAsn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile PheMet, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the oculospanin proteins as needed.

3. Oculospanin Antigen Binding Proteins

The invention provides antigen binding proteins that bind tooculospanin. By “antigen binding protein” as used herein is meant aprotein that specifically binds a specified antigen; the antigen in thepresent invention is human oculospanin.

The antigen binding proteins of the invention specifically bind to humanoculospanin. “Specifically binds” as used herein means the equilibriumdissociation constant is <10⁻⁷ to 10⁻¹⁰ M, more preferably <10⁻⁸ to<10⁻¹⁰ M, even more preferably <10⁻⁹ to <10⁻¹⁰ M. In a specificembodiment, the antigen binding protein binds to the human oculospaninhaving the amino acid sequence of SEQ ID NOs:2 and 4.

3A. Antibodies as Antigen Binding Proteins

In one embodiment, the present invention provides antigen bindingproteins that are antibodies, including, but not limited to, monoclonalantibodies, bispecific antibodies, minibodies, domain antibodies,synthetic antibodies (sometimes referred to herein as “antibodymimetics”), chimeric antibodies, humanized antibodies, antibody fusions(sometimes referred to as “antibody conjugates”), and fragments of each,respectively.

3 A i) Antibody Structures

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). The amino-terminalportion of each chain includes a variable region of about 100 to 110 ormore amino acids primarily responsible for antigen recognition. Each ofthe heavy chains and light chains has a repeat structure, in which anamino acid sequence formed of about 110 residues are conserved, thisconstitutes a basic unit (hereinafter referred to as a “domain”) of thethree dimensional structure of IgG. The heavy chain and light chainconstitute 4 and 2 independent continuous domains, respectively. In boththe heavy chain and the light chain, the variation in the amino terminaldomain between different antibodies is greater than the variation in theother domains. This domain is called a variable domain (hereinafterreferred to as a “V domain”). At the amino terminus of IgG, the Vdomains of the heavy chain and light chain are complementarilyassociated to form a variable region.

According to the results of X-ray crystallography, a domain has alongitudinal cylindrical structure in which two layers of antiparallelβ-sheets each formed of 3 to 5β chains are superposed. In the variableregion, three loops are gathered for each of the V domains of the heavychain and light chain to form an antigen-binding site. Each of the loopsis referred to as a complementarity-determining region (hereinafterreferred to as a “CDR”), in which the variation in the amino acidsequence is most significant. The portions other than the CDRs of thevariable region generally play a role in supporting the structure of theCDR, and are thus called the “framework”. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al.). Furthermore,the frameworks are classified into a plurality of subgroups based oncommon features of the amino acid sequences. Furthermore, it was foundthat there is a corresponding framework between a human and a mouse.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. The constant domain has asequence intrinsic to each animal species. For example, the constantregion of mouse IgG differs from that of human IgG. Therefore, mouse IgGis recognized as a foreign substance by the human immune system. As aresult, a human anti-mouse antibody response (hereinafter referred to as“HAMA”) is raised (see Schroff et al., Cancer Res., 45, 879-85 (1985).Because of this, mouse antibodies are generally not administeredrepeatedly to a human subject.

Human light chains are classified as kappa and lambda light chains.Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. IgG has several subclasses, including, but not limited toIgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but notlimited to, IgM1 and IgM2. The antibodies of the invention may be of anytype including IgM, IgG (including IgG1, IgG2, IgG3, IgG4), IgD, IgA, orIgE antibody. In specific embodiment, the antigen binding protein is anIgG type antibody, with specific embodiments including antibodies withIgG1 sequences All subclasses are contemplated within the presentinvention.

Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about twelve (12) or more amino acids, withthe heavy chain also including a “D” region of about ten (10) more aminoacids. See, generally, Paul, W., ed., 1989, Fundamental Immunology Ch.7, 2nd ed. Raven Press, N.Y. The variable regions of each light/heavychain pair form the antibody binding site.

The variable regions of the heavy and light chains typically exhibit thesame general structure of relatively conserved framework regions (FR)joined by three hypervariable regions, also called complementaritydetermining regions or CDRs. The CDRs are the hypervariable regions ofan antibody (or antigen binding protein, as outlined herein), that areresponsible for antigen recognition and binding. The CDRs from the twochains of each pair are aligned by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest. Chothia et al., 1987, J. Mol. Biol. 196:901-917; Chothia etal., 1989, Nature 342:878-883.

CDRs constitute the major surface contact points for antigen binding.See, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. Further,CDR3 of the light chain and, especially, CDR3 of the heavy chain mayconstitute the most important determinants in antigen binding within thelight and heavy chain variable regions. See, e.g., Chothia and Lesk,1987, supra; Desiderio et al., 2001, J. Mol. Biol. 310:603-615; Xu andDavis, 2000, Immunity 13:37-45; Desmyter et al., 2001, J. Biol. Chem.276:26285-26290; and Muyldermans, 2001, J. Biotechnol. 74:277-302. Insome antibodies, the heavy chain CDR3 appears to constitute the majorarea of contact between the antigen and the antibody. Desmyter et al.,2001, supra. In vitro selection schemes in which CDR3 alone is variedcan be used to vary the binding properties of an antibody. Muyldermans,2001, supra; Desiderio et al., 2001, supra.

Naturally occurring antibodies typically include a signal sequence,which directs the antibody into the cellular pathway for proteinsecretion and which is not present in the mature antibody. Apolynucleotide encoding an antibody of the invention may encode anaturally occurring signal sequence or a heterologous signal sequence asdescribed below.

3 A i) a) Chimeric and Humanized Antibodies

In some embodiments, however, the scaffold components can be a mixturefrom different species. As such, if the antigen binding protein is anantibody, such antibody may be a chimeric antibody and/or a humanizedantibody. In general, both “chimeric antibodies” and “humanizedantibodies” refer to antibodies that combine regions from more than onespecies. For example, “chimeric antibodies” traditionally comprisevariable region(s) from a mouse (or rat, in some cases) and the constantregion(s) from a human. “Humanized antibodies” generally refer tonon-human antibodies that have had the variable-domain framework regionsswapped for sequences found in human antibodies. Generally, in ahumanized antibody, the entire antibody, except the CDRs, is encoded bya polynucleotide of human origin or is identical to such an antibodyexcept within its CDRs. The CDRs, some or all of which are encoded bynucleic acids originating in a non-human organism, are grafted into thebeta-sheet framework of a human antibody variable region to create anantibody, the specificity of which is determined by the engrafted CDRs.The creation of such antibodies is described in, e.g., WO 92/11018,Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science239:1534-1536. Humanized antibodies can also be generated using micewith a genetically engineered immune system. Roque et al., 2004,Biotechnol. Prog. 20:639-654.

In one embodiment, the invention provides antibodies which have one ormore CDRs from the murine antibody produced by the mouse hybridomaO3B8-2C9-4F3, deposited at the International Patent Organism Depository,National Institute of Advanced Industrial Science and Technology,Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken305-8566, JAPAN, deposit number FERM BP-08627. That is, any combinationof heavy and light chain CDRs, including CDR1; CDR2 and CDR3 from thelight and heavy chains, from O3B8-2C9-4F3 can be used. In addition,amino acid variants of the murine CDRs that retain substantially thesame activity as the parent antibody, e.g. binding to oculospanin andinduction of cytoxicity, are also included, as is outlined above forvariantst.

From studies of the structural features of IgG, a method of preparing ahumanized antibody has been conceived as described below.

Initially, a chimeric antibody was proposed in which the variable regionof an antibody derived from a mouse is connected to a constant regionderived from a human (see Proc. Natl. Acad. Sci. U.S.A. 81, 6851-6855,(1984)). However, such a chimeric antibody still contains many non-humanamino acid residues. Therefore, when the chimeric antibody isadministered over a long period of time, a HAMA response may possibly beinduced (see Begent et al., Br. J. Cancer, 62, 487, (1990)).

As a method for further reducing the amino acid residues derived from anon-human mammalian source, which may possibly cause a HAMA response inhumans, a method of integrating only the CDR portion into ahuman-derived antibody was proposed (see Nature, 321, 522-525, (1986)).However, to maintain immunoglobulin activity against an antigen,transplantation of the CDR alone was generally insufficient.

Chothia et al., found the following based on data obtained by X-raycrystallography in 1987:

(i) in the amino acid sequence of the CDR, there is a site which bindsdirectly to an antigen and a site responsible for maintaining thestructure of the CDR itself, and the three dimensional structures of theCDR that can be adopted are classified into a plurality of typicalpatterns (canonical structures); and

(ii) the classes of canonical structures are determined not only by theCDR, but also by the type of amino acid present in a specific site ofthe framework portion (see J. Mol. Biol., 196, 901-917, (1987)).

Based on these findings, it was suggested that when the CDRtransplantation method is employed, amino acid residues of a part of theframework must be transplanted into a human antibody in addition to thesequence of the CDR (see Japanese National Publication of InternationalPatent Application No. 4-502408).

Generally, a non-human mammalian-derived antibody from which the CDR isto be transplanted is defined as a “donor”, whereas the human antibodyinto which the CDR is transplanted is defined as an “acceptor”. Thepresent invention follows these definitions.

A point which should be considered in carrying out the CDRtransplantation is that the activity of the immunoglobulin molecule ismaintained by preserving the CDR structure as much as possible. Toachieve this, attention must be paid to the following two points:

(i) which subgroup of antibodies the acceptor is selected from; and

(ii) which amino acid residue is selected from the framework of thedonor.

Queen et al. proposed a design method for transplanting an amino acidresidue into an acceptor together with the CDR sequence when the aminoacid residue of the framework of the donor corresponds to at least oneof the following references (see Japanese National Publication ofInternational Patent Application No. 4-502408).

(a) the amino acid is rarely present at the position within theframework of an acceptor, whereas the corresponding amino acid of adonor is usually present at the equivalent position;

(b) the amino acid is present near one of the CDRs; and

it is predicted that the amino acid has a side chain atom within about 3angstroms from the CDR in its three dimensional immunoglobulin model andthat the side main atom can interact with an antigen or the CDR of ahumanized antibody.

3. A i) b) Bispecific Antibodies

In one embodiment, the oculospanin antigen binding protein is amultispecific antibody, and notably a bispecific antibody, alsosometimes referred to as “diabodies”. These are antibodies that bind totwo (or more) different antigens. Diabodies can be manufactured in avariety of ways known in the art (Holliger and Winter, 1993, CurrentOpinion Biotechnol. 4:446-449), e.g., prepared chemically or from hybridhybridomas.

3. A i) c) Minibodies

In one embodiment, the oculospanin antigen binding protein is aminibody. Minibodies are minimized antibody-like proteins comprising ascFv joined to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061.

3. A i) d) Domain Antibodies

In one embodiment, the oculospanin antigen binding protein is a domainantibody; see for example U.S. Pat. No. 6,248,516. Domain antibodies(dAbs) are functional binding domains of antibodies, corresponding tothe variable regions of either the heavy (VH) or light (VL) chains ofhuman antibodies dABs have a molecular weight of approximately 13 kDa,or less than one-tenth the size of a full antibody. dABs are wellexpressed in a variety of hosts including bacterial, yeast, andmammalian cell systems. In addition, dAbs are highly stable and retainactivity even after being subjected to harsh conditions, such asfreeze-drying or heat denaturation. See, for example, U.S. Pat. Nos.6,291,158; 6,582,915; 6,593,081; 6,172,197; US Serial No. 2004/0110941;European Patent 0368684; U.S. Pat. No. 6,696,245, WO04/058821,WO04/003019 and WO03/002609.

3. A i) e) Antibody Fragments

In one embodiment, the oculospanin antigen binding protein is anantibody fragment, that is a fragment of any of the antibodies outlinedherein that retain binding specificity to oculospanin.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546) which consists of a singlevariable, (v) isolated CDR regions, (vi) F(ab′)₂ fragments, a bivalentfragment comprising two linked Fab fragments (vii) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., 1988, Science 242:423-426, Huston etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883), (viii)bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”or “triabodies”, multivalent or multispecific fragments constructed bygene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479;WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90:6444-6448). The antibody fragments may be modified. For example, themolecules may be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245).

3. A i) f) Human Antibodies

In one embodiment, the oculospanin antigen binding protein is a fullyhuman antibody. “Fully human antibody” or “complete human antibody”refers to a human antibody having only the gene sequence of an antibodyderived from a human chromosome. The anti-human oculospanin completehuman antibody can be obtained by a method using a humanantibody-producing mouse having a human chromosome fragment containingthe genes for a heavy chain and light chain of a human antibody [seeTomizuka, K. et al., Nature Genetics, 16, p. 133-143, 1997; Kuroiwa, Y.et al., Nuc. Acids Res., 26, p. 3447-3448, 1998; Yoshida, H. et al.,Animal Cell Technology: Basic and Applied Aspects vol. 10, p. 69-73(Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer AcademicPublishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA, 97,722-727, 2000, etc.] or obtained by a method for obtaining a humanantibody derived from a phage display selected from a human antibodylibrary [see Wormstone, I. M. et al., Investigative Ophthalmology &Visual Science. 43(7), p. 2301-8, 2002; Carmen, S. et al., Briefings inFunctional Genomics and Proteomics, 1 (2), p. 189-203, 2002;Siriwardena, D. et al., Ophthalmology, 109(3), p. 427-431, 2002, etc.]

In one embodiment, the oculospanin antigen binding protein is anantibody analog, sometimes referred to as “synthetic antibodies.” Forexample, a variety of recent work utilizes either alternative proteinscaffolds or artificial scaffolds with grafted CDRs. Such scaffoldsinclude, but are not limited to, mutations introduced to stabilize thethree-dimensional structure of the binding protein as well as whollysynthetic scaffolds consisting for example of biocompatible polymers.See, for example, Korndorfer et al., 2003, Proteins: Structure,Function, and Bioinformatics, Volume 53, Issue 1:121-129. Roque et al.,2004, Biotechnol. Prog. 20:639-654. In addition, peptide antibodymimetics (“PAMs) can be used, as well as work based on antibody mimeticsutilizing fibronection components as a scaffold.

3. A i) h) Antibody Conjugates

In one embodiment, the oculospanin antigen binding protein is anantibody fusion protein (sometimes referred to herein as an “antibodyconjugate”). The conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antigen binding protein (see the discussion on covalentmodifications of the antigen binding proteins) and on the conjugatepartner. For example linkers are known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see, 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antigen binding proteins, or binding of a radionuclideto a chelating agent that has been covalently attached to the antigenbinding protein. Additional embodiments utilize calicheamicin,auristatins, geldanamycin and maytansine.

Additional fusion proteins are discussed below with particular referenceto screening assays.

3. A ii) Covalent modifications of Antigen Binding Proteins such asAntibodies

Covalent modifications of antigen binding proteins, as well as theoculospanin proteins themselves, are included within the scope of thisinvention, and are generally, but not always, done post-translationally.For example, several types of covalent modifications of the antigenbinding protein are introduced into the molecule by reacting specificamino acid residues of the antigen binding protein with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantigen binding proteins to a water-insoluble support matrix or surfacefor use in a variety of methods, in addition to methods described below.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the antigen binding proteinincluded within the scope of this invention comprises altering theglycosylation pattern of the protein. As is known in the art,glycosylation patterns can depend on both the sequence of the protein(e.g., the presence or absence of particular glycosylation amino acidresidues, discussed below), or the host cell or organism in which theprotein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence is preferablyaltered through changes at the DNA level, particularly by mutating theDNA encoding the target polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antigen binding proteincomprises linking the antigen binding protein to variousnonproteinaceous polymers, including, but not limited to, variouspolyols such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, asis known in the art, amino acid substitutions may be made in variouspositions within the antigen binding protein to facilitate the additionof polymers such as PEG.

3. A iii) Labeled Antibodies

In some embodiments, the covalent modification of the antigen bindingproteins of the invention comprises the addition of one or more labels.In some cases, these are considered antibody fusions.

The term “labelling group” means any detectable label. In someembodiments, the labelling group is coupled to the antigen bindingprotein via spacer arms of various lengths to reduce potential sterichindrance. Various methods for labelling proteins are known in the artand may be used in performing the present invention.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labelling groupis coupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabelling proteins are known in the art and may be used in performingthe present invention.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in Molecular Probes Handbookby Richard P. Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558). All of the above-cited references are expressly incorporatedherein by reference.

4. Nucleic Acids Encoding Oculospanin Antigen Binding Proteins

The invention provides nucleic acids that encode oculospanin andoculospanin antigen binding proteins, as outlined herein (in addition tonucleic acids as candidate agents, as described below). It should benoted that the following discussion can also apply to nucleic acidsencoding oculospanin antigen binding proteins, nucleic acids used ascandidate agents (e.g. antisense molecules), or to nucleic acidsencoding proteinaceous candidate agents, as described below.

As will be appreciated by those in the art, the depiction of a singlestrand (“Watson”) also defines the sequence of the other strand(“Crick”); thus the nucleic acid sequences depicted herein (e.g. SEQ IDNO:1 and SEQ ID NO:4) also include the complement of these sequences. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,described below, originally formed in vitro, in general, by themanipulation of nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated oculospanin nucleic acid, in a linearform, or an expression vector formed in vitro by ligating DNA moleculesthat are not normally joined, are both considered recombinant for thepurposes of this invention. It is understood that once a recombinantnucleic acid is made and reintroduced into a host cell or organism, itwill replicate non-recombinantly, i.e., using the in vivo cellularmachinery of the host cell rather than in vitro manipulations; however,such nucleic acids, once produced recombinantly, although subsequentlyreplicated non-recombinantly, are still considered recombinant for thepurposes of the invention.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the oculospanin antigen binding proteins andoculospanin proteins of the present invention. Thus, having identified aparticular amino acid sequence those skilled in the art could make anynumber of different nucleic acids, by simply modifying the sequence ofone or more codons in a way that does not change the amino acid sequenceof the encoded protein.

In general, oculospanin nucleic acids hybridize to the sequencesdepicted in SEQ ID NO:1 and SEQ ID NO:4 under high stringencyconditions. The term “hybridizes under stringent conditions” refers tohybridization which is performed at 68° C. in a commercially availablehybridization solution, namely ExpressHyb (manufactured by Clontech), orhybridization which is performed at 68° C. in the presence of NaCl at0.7 to 1.0 M using a filter having DNA immobilized thereon, followed bywashing at 68° C. with 0.1 to 2×SSC solution (1×SSC solution contains150 mM NaCl and 15 mM sodium citrate), resulting in hybridization. Theabove term also includes hybridization under conditions equivalent tothose above.

In some embodiments, the oculospanin proteins (or antigen bindingproteins, in some instances) are isolated proteins or substantially pureproteins. An “isolated” protein is unaccompanied by at least some of thematerial with which it is normally associated in its natural state,preferably constituting at least about 5%, more preferably at leastabout 50% by weight of the total protein in a given sample. A“substantially pure” protein comprises at least about 75% by weight ofthe total protein, with at least about 80% being specific, and at leastabout 90% being particularly specific. The definition includes theproduction of a protein from one organism in a different organism orhost cell. Alternatively, the protein may be made at a significantlyhigher concentration than is normally seen, through the use of aninducible promoter or high expression promoter, such that the protein ismade at increased concentration levels. Thus, a “recombinant protein” isa protein made using recombinant techniques, i.e., through theexpression of a recombinant nucleic acid as described herein. Methodsand techniques for the production of recombinant proteins are well knownin the art and described below in the “nucleic acid” section.

4 A. Preparation of Oculospanin Proteins

There are a variety of techniques useful in obtaining oculospaninnucleic acids, as is well known in the art. Generally, probe polymerasechain reaction (PCR) primer sequences, based on the sequences of SEQ IDNO:1 and SEQ ID NO:4, may be used to clone expression vectors, as wellas find other related oculospanin proteins from other organisms asneeded. As will be appreciated by those in the art, particularly usefulprobe and/or PCR primer sequences include the unique areas of theoculospanin nucleic acid sequence. As is generally known in the art,preferred PCR primers are from about 15 to about 35 nucleotides inlength, with from about 20 to about 30 being preferred, and may containinosine as needed. The conditions for the PCR reaction are well known inthe art.

Once the oculospanin protein is amplified, it can be cloned and, ifnecessary, its constituent parts recombined to form the entireoculospanin nucleic acid. Once isolated from its natural source, e.g.,contained within a plasmid or other vector or excised therefrom as alinear nucleic acid segment, the recombinant oculospanin nucleic acidcan be expressed as outlined below.

4 B. Nucleic Acids Encoding Oculospanin Antigen Binding Proteins Such asAntibodies

In the case of antigen binding proteins such as antibodies, nucleicacids, particularly DNA, encoding heavy chains and/or light chains ofthe anti human oculospanin monoclonal antibodies (or other types ofantibodies, as outlined herein) of the present invention can be obtainedby preparing mRNA from a hybridoma cell producing the anti-humanoculospanin monoclonal antibody, converting the mRNA into cDNA usingreverse transcriptase, and isolating each DNA encoding the heavy chainor light chain of the antibody.

In extracting mRNA, the guanidine thiocyanate—hot phenol method, andguanidine thiocyanate guanidine—hydrochloride method may be employed;however, the guanidine thiocyanate cesium chloride method is alsosuitable. Preparation of mRNA from a cell is performed by firstpreparing total RNA and purifying the mRNA using a poly(a)⁺ RNApurification carrier such as oligo (dT) cellulose or oligo (dT) latexbeads or directly purifying mRNA from a cell lysate by use of thecarrier. For preparing total RNA, use may be made of the alkalinesucrose density-gradient centrifugation method [see Dougherty, W. G. andHiebert, E. (1980) Virology 101, 466-474], the guanidinethiocyanate-phenol method, the guanidine thiocyanate-trifluoro-cesiummethod, and the phenol SDS method and the like; however, the methodusing guanidine thiocyanate and cesium chloride is also suitable [seeChirgwin, J. M., et al. (1979) Biochemistry 18, 5294-5299].

After a single-stranded cDNA is synthesized by a reverse transcriptasereaction using the poly(a)+RNA obtained as mentioned above as atemplate, double-stranded cDNA can be synthesized from thesingle-stranded cDNA. This method may be the S1 nuclease method [seeEfstratiadis, A., et al. (1976) Cell, 7, 279-288], the Gubler/Hoffmannmethod [see Gubler, U. and Hoffman, B. J. (1983) Gene 25, 263-269], theOkayama/Berg method [see Okayama, H. and Berg, P. (1982) Mol. Cell.Biol. 2, 161-170] or others; however, suitably used in the presentinvention is the so-called RT-PCR method in which a polymerase chainreaction (hereinafter referred to as a “PCR”) [see Saiki, R. K., et al.(1988) Science 239, 487-49] is performed using a single-stranded cDNA asa template.

The double-stranded cDNA thus obtained is integrated into a cloningvector to obtain a recombinant vector, which is then introduced into amicroorganism, such as Escherichia coli, to form a transformant. Thetransformant can be selected by using tetracycline resistance orampicillin resistance as a marker. Escherichia coli can be transformedby the Hanahan method [see Hanahan, D. (1983) J. Mol. Biol. 166,557-580], more specifically, by preparing a competent cell in thepresence of calcium chloride, magnesium chloride or rubidium chloride,and adding the recombinant DNA vector to the competent cell. Note thatwhen a plasmid is used as a vector, the plasmid must have any one of thedrug resistance genes as mentioned above. Needless to say, a cloningvector other than a plasmid, such as a lambda group phage, may be used.

As a method of selecting a strain having a cDNA, which encodes each ofthe subunits of a desired anti-human oculospanin monoclonal antibodyfrom the transformant strain obtained above, any of the methodsdescribed below can be employed. When a desired cDNA is specificallyamplified by the RT-PCR method, such operation of the method can beskipped.

4 C. Methods of Producing Proteins Including Oculospanin and AntigenBinding Proteins

The present invention also provides expression systems and constructs inthe form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one nucleic acid, as outlined herein.In addition, the invention provides host cells comprising suchexpression systems or constructs. As outlined herein, these nucleicacids can encode oculospanin proteins, oculospanin antigen bindingproteins such as anti-oculospanin antibodies, or candidate agents, asdefined below.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the oculospaninantigen binding protein coding sequence; the oligonucleotide sequenceencodes polyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the oculospanin antigen binding proteinfrom the host cell. Affinity purification can be accomplished, forexample, by column chromatography using antibodies against the tag as anaffinity matrix. Optionally, the tag can subsequently be removed fromthe purified oculospanin antigen binding protein by various means suchas using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein antibody that binds to oculospanin polypeptide.As a result, increased quantities of a polypeptide such as anoculospanin antigen binding protein are synthesized from the amplifiedDNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the oculospanin antigen binding protein. Promotersare untranscribed sequences located upstream (i.e., 5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encoding heavychain or light chain comprising an oculospanin antigen binding proteinof the invention by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the desired promoter sequenceinto the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell, 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising anoculospanin antigen binding protein of the invention by highereukaryotes. Enhancers are cis-acting elements of DNA, usually about10-300 bp in length, that act on the promoter to increase transcription.Enhancers are relatively orientation and position independent, havingbeen found at positions both 5′ and 3′ to the transcription unit.Several enhancer sequences available from mammalian genes are known(e.g., globin, elastase, albumin, alpha-feto-protein and insulin).Typically, however, an enhancer from a virus is used. The SV40 enhancer,the cytomegalovirus early promoter enhancer, the polyoma enhancer, andadenovirus enhancers known in the art are exemplary enhancing elementsfor the activation of eukaryotic promoters. While an enhancer may bepositioned in the vector either 5′ or 3′ to a coding sequence, it istypically located at a site 5′ from the promoter. A sequence encoding anappropriate native or heterologous signal sequence (leader sequence orsignal peptide) can be incorporated into an expression vector, topromote extracellular secretion of the antibody. The choice of signalpeptide or leader depends on the type of host cells in which theantibody is to be produced, and a heterologous signal sequence canreplace the native signal sequence. Examples of signal peptides that arefunctional in mammalian host cells include the following: the signalsequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195;the signal sequence for interleukin-2 receptor described in Cosman etal., 1984, Nature 312:768; the interleukin-4 receptor signal peptidedescribed in EP Patent No. 0367 566; the type I interleukin-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; the type IIinterleukin-1 receptor signal peptide described in EP Patent No. 0 460846.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector as outlined above. Suchvectors may or may not contain all of the desired flanking sequences.Where one or more of the flanking sequences described herein are notalready present in the vector, they may be individually obtained andligated into the vector. Methods used for obtaining each of the flankingsequences are well known to one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding all or part of the oculospanin antigen binding protein oroculospanin protein has been inserted into the proper site of the vector(or plurality of vectors, as the case may be), the completed vector maybe inserted into a suitable host cell for amplification and/orpolypeptide expression. The transformation of an expression vector foran oculospanin antigen binding protein into a selected host cell may beaccomplished by well known methods including transfection, infection,calcium phosphate co-precipitation, electroporation, microinjection,lipofection, DEAE-dextran mediated transfection, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan, and are set forth, for example, inSambrook et al., 2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes anoculospanin antigen binding protein that can subsequently be collectedfrom the culture medium (if the host cell secretes it into the medium)or directly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines may be selected throughdetermining which cell lines have high expression levels andconstitutively produce antigen binding proteins with oculospanin bindingproperties. In another embodiment, a cell line from the B cell lineagethat does not make its own antibody but has a capacity to make andsecrete a heterologous antibody can be selected.

5. Use of Oculospanin Antigen Binding Proteins for Diagnostic,Therapeutic and Screening Purposes

In the specification of the present invention, a compound having acancer therapeutic effect is a compound having an activity insuppressing cancer growth and/or an activity of reducing cancer. In thespecification of the present invention, the terms “cancer” and “tumor”have the same meaning. The term “canceration of a cell” used hereinrefers to the abnormal proliferation of cells, which is caused by theirlack of sensitivity to contact inhibition and their scaffoldindependent-proliferation. A cell exhibiting such abnormal proliferationis referred to as a “cancer cell”. In the specification, a proteinhaving the same function as that of human oculospanin, such ascanceration activity, is also referred to as a “human oculospanin”. Notethat the term “oncogene” as used in the present invention includes aprecancerous gene and a proto-oncogene other than the oncogene.

The present invention provides agents that confer and/or inducecytotoxicity to cells expressing oculospanin. The term “cytotoxicity”used herein refers to both cell death and toxicity resulting in cellstasis, e.g. a loss of the ability to grow, as well as to apoptosis.Therefore, cytotoxicity includes not only externally inflicted directdamage, but also various structural and functional changes that mayoccur within a cell, which include DNA cleavage, dimerization of bases,chromosomal cleavage, malfunction of cellular mitotic apparatus, and areduction in enzymatic activities. The term “cytotoxic activity” usedherein refers to any activity that causes cytotoxicity, as mentionedabove.

The correlation of the connection between oculospanin (includingoculospanin levels in cancerous cells) allows a number of utilities,including diagnostic methods and kits, therapeutic uses, including incancer, and screening technologies for modulation of oculospaninactivity.

5 A. Diagnosis Utilizing Oculospanin and Oculospanin Antigen BindingProteins

As is described herein, oculospanin has been shown to be over expressedin cancer cells, particularly melanoma cells. Accordingly, the inventionprovides a number of diagnostic methods and kits, based either onprotein or nucleic acid detection, for the detection of cancer.

5 A i). Confirmation of Specific Expression of the Human OculospaninGene for Diagnosis

As a result of analyzing expression levels of the human oculospanin genein various types of human cells, it was found that the gene is expressedat a significantly higher expression level in melanocytes compared toother tissues. Furthermore, the present inventors found that the levelof expression of the human oculospanin gene in melanoma is significantlyhigher than in normal melanocytes. To explain more specifically, theyfound the following: when the level of expression of human oculospaninin melanocytes, lymphoblasts and glia cells, and epithelial cells iscompared, the expression level in melanocytes is found to besignificantly higher. Furthermore, when the level of expression of humanoculospanin in normal skin cells is compared to that in melanoma, theexpression level is significantly higher in the melanoma. From thesefindings, it can be concluded that human oculospanin may be involved incanceration of cells and/or in proliferation of cancer cells. Thissuggests that the canceration state and/or proliferation state of cancercells caused by excessive expression of human oculospanin can bedetermined by measuring the level of expression of human oculospanin inindividual cells and/or tissues. An example of such cancer is skincancer, in particular, melanoma. However, this finding is applicable tocancers other than skin cancer, provided that human oculospanin isexpressed in the cancer at a significantly higher level than in othertissues.

The nucleotide sequence of the open reading frame (ORF) of the humanoculospanin gene is represented by Sequence ID No. 1 of the sequencelisting and the amino acid sequence thereof is represented by SequenceID No. 2. Furthermore, cDNA of the human oculospanin gene has beenregistered with GenBank as Homo sapiens oculospanin (OCSP) mRNA underAccession No. NM_(—)031945. The cDNA nucleotide sequence registered atGenBank is represented by Sequence ID No. 3 of the sequence listing. TheORF is represented by nucleotide Nos. 65 to 1129 of Sequence ID No. 3.Furthermore, the amino acid sequence of human oculospanin registered atGenBank is represented by Sequence ID No. 4 of the sequence listing.There is a single amino acid difference between SEQ ID NO:2 and SEQ IDNO:4. A protein comprising an amino acid sequence having one or severalamino acids replaced, deleted from or added to the amino acid sequenceof human oculospanin and exhibiting the same biological activity as thatof human oculospanin is also included herein as a human oculospanin.

5. B Diagnostic Assays Using Nucleic Acids

As outlined herein, the present invention provides diagnostic assays andkits based on nucleic acids and/or proteins.

Human oculospanin, since it is highly expressed in cancer cells,especially, melanoma, is thought to be involved in canceration of cells,particularly skin cells, and/or proliferation of cancer cells.

Thus the present invention provides methods of detecting cancer, or apredisposition or propensity to get cancer, in patient samples.

The term “sample” or “specimen” refers to a sample taken from a testsubject or a clinical specimen, and includes samples of tissues,excrement or the like, such as samples of blood, body fluids, prostategland, testes, penis, bladder, kidney, oral cavity, pharynx, lip,tongue, gingival, nasopharynx, esophagus, stomach, small intestine,large intestine, colon, liver, gall bladder, pancreas, nose, lung, bone,soft tissue, skin, breast, uterus, ovary, brain, thyroid, lymph node,muscle, and adipose tissue. In the present invention, skin and lymphnode are preferred tissue samples.

In one embodiment, the invention provides methods of detecting cancerusing the level of expression of the human oculospanin gene.

In a first embodiment, the method utilizes the following steps 1) to 4):

1) a step of extracting a total RNA fraction from a specimen taken froma test subject;

2) a step of extracting a total RNA fraction from a specimen taken froma healthy person;

3) a step of measuring the level of expression of the human oculospaningene in the total RNA fractions according to steps 1) and 2); and

4) a step of analyzing the difference in the level of expression of thegene between the total RNA fraction derived from steps 1) and 2),measured in step 3) and thereby detecting cancer of the test subject ofstep 1).

In one embodiment, the steps are as follows. Step 1 comprises extractinga total RNA fraction from a specimen taken from a test subject.

In extracting the total RNA fraction from a specimen, human tissue isobtained by an appropriate method satisfying the ethical standards forexperimentation. The tissue obtained is dissolved directly in an RNAextraction solvent (containing a ribonuclease inhibitor, such asphenol). Alternatively, cells of the tissue obtained are collected byabrading them using a scraper so as not to break the cells, or gentlyextracting them from the tissue using a proteolytic enzyme such astrypsin, and then immediately subjecting the cells to an RNA extractionstep.

Examples of RNA extraction methods that may be used include: guanidinethiocyanate/cesium chloride ultracentrifugation methods, guanidinethiocyanate/hot phenol methods, guanidine hydrochloride methods, andacidic guanidine thiocyanate/phenol/chloroform methods (Chomczynski, P.and Sacci, N., Anal. Biochem. (1987), 162, 156-159). Of these, acidicguanidine thiocyanate/phenol/chloroform methods are particularlysuitable. Alternatively, a commercially available RNA extractionreagent, such as ISOGEN (manufactured by Nippon Gene Co., Ltd.) orTRIZOL reagent (manufactured by Gibco BRL) may be used in accordancewith the protocol provided with the reagent.

From the total RNA fraction obtained, if necessary, it is preferred thatmRNA alone is purified and used. Any suitable purification method can beused. For example, mRNA can be purified by adsorbing mRNA onto abiotinylated oligo. (dT) probe, attaching the mRNA to paramagneticparticles having streptavidin immobilized thereon via binding of biotinto streptavidin, washing the particles, and eluting mRNA. Alternatively,mRNA may be purified by adsorbing mRNA onto an oligo (dT) cellulosecolumn and eluting the mRNA therefrom. However, an mRNA purificationstep is not essential in methods of the present invention. Provided thatexpression of a desired polynucleotide can be detected, a total RNAfraction may be used, as can be done in the later steps.

Step 2 comprises a control step, e.g. extracting a total RNA fractionfrom a specimen taken from a healthy person. In the present invention, ahealthy person means a person who does not have cancer. Thedetermination as to whether or not a person is healthy can be made bymeasuring the concentration of human oculospanin and determining whetheror not the concentration value measured falls within a predeterminedrange for a healthy person. Alternatively, the correlation between theexpression level of human oculospanin and the degree of cancer formationcan be investigated in advance, and then, determination of whether ornot a test subject is a healthy person can be made by measuring theexpression level of human oculospanin in a specimen taken from the testsubject. The preparation of a total RNA fraction from a healthy personcan be performed in the same manner as described in Step 1) above.

It should be noted that in some instances, for example when the level ofoculospanin expression from a particular tissue, patient or sample isalready known, it is not necessary to determine the level of oculospaninexpression from a “normal” or “healthy” sample.

Step 3 can be done by measuring the level of expression of the humanoculospanin gene in a total RNA fraction according to steps 1) and 2).

The level of expression of the human oculospanin gene is represented bythe expression level of a polynucleotide that can hybridize with apolynucleotide which comprises the nucleotide sequence represented bySequence ID No. 1 of the sequence listing or a polynucleotide whichcomprises a nucleotide sequence complementary to the nucleotide sequencerepresented by Sequence ID No. 1 of the sequence listing, understringent conditions.

It should be noted that in some cases, it is desirable to use the entireoculospanin gene in the expression analysis; in other embodiments, suchas in the use of gene arrays, as described below, the nucleic acidprobes used to test for the presence of oculospanin nucleic acid can befragments of the full length gene. Thus, for example, fragments ofoculospanin nucleic acid can be used as the probe to determine theexpression levels. In general, the probes will range from about 8nucleosides to about 100, with from about 10 to 50 being preferred, andfrom about 15 to 30 being particularly preferred. As will be appreciatedby those in the art, the length of the probes used is generallysufficient to confer specificity. In addition, it should be appreciatedthat either the coding (“Watson”) strand or the non-coding strand(“Crick”) can be used, depending on the assay.

As will be appreciated by those in the art, diagnostic assays can be runeither as solution phase assays (homogeneous assays) or as solid phaseassays (heterogeneous assays).

In one embodiment, solution assays are run. In these cases, assaysgenerally rely on probes that bind the oculospanin nucleic acids basedon either the increase or decrease of fluorescence based onhybridization status, or on fluorescence resonance energy transfer(FRET) assays. For example, “molecular beacon” probes contain two labelsand form hairpin loops that are quenched in the absence of targetsequence; upon hybridization, the two labels are separated and a signalis generated. See for example U.S. Pat. Nos. 5,925,517, 6,103,476,6,461,817 and 6,037,130, as well as other PHRI patents and applications,incorporated by reference herein. Similarly, “Hybeacon” probes aresingle-stranded probes that are labeled with a single fluorophore; uponbinding to a complementary nucleic acid, the emission spectra of thelabel is altered and thus detected. FRET assays are done using probesand targets that contain two different labels; upon binding, the labelsbecome spatially close so as to allow energy transfer.

In one embodiment, heterogeneous assays are done using nucleic acidsattached to a solid support for testing specimens for binding and/orquantitation of the oculospanin nucleic acid. By “substrate” or “solidsupport” or other grammatical equivalents herein is meant any materialappropriate for the attachment of capture probes and is amenable to atleast one detection method. As will be appreciated by those in the art,the number of possible substrates is very large. Possible solid supportsinclude, but are not limited to, glass and modified or functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, Teflon, etc.), polysaccharides, nylon or nitrocellulose,resins, silica or silica based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, plastics, ceramics, and avariety of other polymers. In a some embodiments, the solid supportsallow optical detection and do not themselves appreciably fluoresce. Inaddition, as is known the art, the solid support may be coated with anynumber of materials, including polymers, such as dextrans, acrylamides,gelatins, agarose, etc. Exemplary solid supports include silicon, glass,polystyrene and other plastics and acrylics.

Generally the solid support is flat (planar), although as will beappreciated by those in the art, other configurations of solid supportsmay be used as well, including the placement of the probes on the insidesurface of a tube, for flow-through sample analysis to minimize samplevolume.

In one embodiment, the support is a gene chip. A gene chip may be usedon which there is immobilized either an anti-sense oligonucleotide,which is synthesized based on an EST (expressed sequence tag) sequencefrom a database, known sequences (e.g. oculospanin sequences, forexample oculospaninprobes) or an mRNA sequence. In some cases, fulllength genes or complements can be used. Examples of such gene chipsinclude gene chips manufactured by Affymetrix (Lip Shutz, R. J. et al.,Nature Genet. (1999), 21, supplement, 20-24), but are not limitedthereto, and may be prepared based on any known method. When mRNAderived from a human cell is analyzed, a gene chip derived from humansequences is preferably used. For example, the human sequences U95 setor U133 set manufactured by Affymetrix may be used. However, suitablegene chips are not limited to these and a gene chip derived from, forexample, an animal species closely related to a human may be used.

In an alternative embodiment, membrane filters on which there isimmobilized a cDNA or RT-PCR product prepared from total human RNA ortotal RNA, taken from a specific tissue of a human subject, ESTsequences, oculospanin sequences, etc. can be used.

The sample can be a number of things. The cDNA or RT-PCR product can bea clone obtained by performing a reverse transcription reaction and PCRusing a primer prepared based from the oculospanin sequence. The cDNA orRT-PCR product may have been selected previously by use of a subtractionmethod (Diatchenki, L, et al., Proc. Natl, Acad. Sci, USA (1996) 93,6025-6030) or a differential display method (Liang, P., et al., NucleicAcids Res., (1992) 23, 3685-3690) based on total RNA in which theexpression level differs between a person having a tumor and a personhaving no tumor. The array or filter may be one which is commerciallyavailable, such as those provided by IntelliGene (manufactured by TakaraBio). Alternatively, the cDNA or RT-PCR product may be immobilized usinga commercially available spotter such as GMS417 arrayer (manufactured byTakara Bio) to make an array or a filter.

In one embodiment, not a specific mRNA clone but all of the expressedmRNA are labeled and used as a labeled sample that is put on the solidsupport. Crude mRNA (unpurified) may be used as a starting material forpreparing a probe; however, preferably poly (A)⁺ RNA is used which hasbeen purified by the aforementioned method. A method of preparing alabeled probe and a method of detecting and analyzing the probe usingvarious types of immobilized sample are further described as follows.

A biotin-labeled cRNA probe is prepared in accordance with the protocol(Affymetrix's Expression Analysis Technical Manual) provided with theGeneChip manufactured by Affymetrix. Subsequently, hybridization andanalysis is performed to detect and analyze light emitted from adipicacid using an Affymetrix analyzer (GeneChip Fluidics Station 400) inaccordance with the protocol (Expression Analysis Technical Manual)provided with the GeneChip manufactured by Affymetrix.

In order to detect cDNA, a label must be attached to the cDNA when it isprepared from poly (A)⁺ RNA using a reverse transcriptase reaction. Toobtain fluorescently labeled cDNA, d-UTP labeled with a fluorescent dyesuch as Cy3 or Cy5 may be included in the reaction mixture. If poly(A)⁺RNA derived from a melanoma cell and poly (A)⁺ RNA derived from a cellused as a control are labeled with different dyes, then both types ofpoly (A)⁺ RNAs may be used simultaneously in a mixture. When acommercially available array is used, e.g. an array manufactured byTakara Bio Co., Ltd. hybridization and washing are performed inaccordance with the protocol provided and then a fluorescent signal isdetected using a fluorescent signal detector (for example, the GMS418array scanner manufactured by Takara Bio Co., Ltd.) and thereaftersubjected to analysis. The choice of array for use as described hereinis not limited to those which are commercially available. An home-madearray and an array specifically prepared in-house may be used. Inaddition, as noted above, it is possible to use “sandwich” assays,wherein the capture probe on the surface of the solid support binds to afirst domain of the target oculospanin sequence, and a label probehybridizes to a second domain of the target sequence. The label probescan include “Molecular Beacons” and “Hybeacons”, or single strandednucleic acids labeled with fluorophores or other labels as outlinedherein.

When preparing cDNA from poly (A)⁺ RNA by reverse transcription, alabeled sample can be prepared by adding a radioisotope (for example,d-CTP) to the reaction. Hybridization is performed in accordance withcustomary methods. More specifically, hybridization can be performedusing the Atlas system (manufactured by Clontech), which is a microarrayformed using a commercially available filter, after hybridization themicroarray is washed. Thereafter, detection and analysis are performedusing an analyzer (for example, Atlas Image manufactured by Clontech).

In any of the methods, a sample derived from human tissue is hybridizedwith the immobilized samples of the same lot. The probe which is usedcan be charged, but the hybridization conditions used are kept the same.When fluorescently labeled probes are used, if the probes are labeledwith different fluorescent dyes, then probes of different types can beadded simultaneously in the form of a mixture and hybridized with theimmobilized samples. Thereafter, fluorescent intensity can be readsimultaneously (Brown, P. O. et al., Nature Genet., (1999) 21,supplement, p. 33-37).

In addition to the measurement methods mentioned above, there aresubtraction cloning methods (see Experimental Medicine, SupplementaryVolume, New Genetic Engineering Handbook, published by Yodosha Co., Ltd.(1996), p32-35); differential display methods (Basic BiochemicalExperimental Method 4, nucleic acid/gene experiment, II. Applied series,Tokyo Kagakudojin (2001), p125-128); and methods using a reporter gene:chloramphenicol acetyltransferase (such as a pCAT3-Basic vectormanufactured by Promega), β-galactosidase (such as a pβgal-Basic vectormanufactured by Promega), secreted alkaline phosphotase (such aspSEAP2-Basic manufactured by Clontech); or green-fluorescent protein(such as pEGFP-1 manufactured by Clontech). However, the choice ofmeasurement method is not limited to these methods.

Using any of the methods described above, The difference in the level ofexpression of human oculospanin between a specimen derived from ahealthy person and a specimen derived from a test subject is analyzed.If a specimen shows a significantly high expression level of humanoculospanin, it is determined that the possibility of having cancer,particularly skin cancer, and more particularly melanoma, is high, thatis, cancer can be detected. The term “significantly high expressionlevel” refers to the case where, when analysis is performed by usingGeneChip (manufactured by Affymetrix) and microarray Suite Ver. 3.0(manufactured by Affymetrix), an average difference value of a genederived from a melanoma cell is significantly high compared to that of anormal melanocyte.

5 C Protein Diagnostic Assays

Alternatively, the level of expression of human oculospanin is measured,and then assessed to determine whether or not the measured concentrationvalue falls within a predetermined range for a healthy person. If thevalue exceeds the range, the subject has cancer. The diagnosis of cancercan be made in this manner. Otherwise, the correlation between the levelof expression of the human oculospanin gene and the degree of cancerformation in a healthy person is previously investigated, and then, theexpression level of human oculospanin gene of a specimen taken from thetest subject is measured. Also, in this manner, whether or not a testsubject is a healthy person or not can be determined.

In addition to the nucleic acid diagnostic methods described herein,diagnosis can be done using protein expression assays as well. Ingeneral, this is done by measuring the level of expression of humanoculospanin protein in a specimen taken from a subject and comparing thelevel to the level of expression in a healthy subject. Again, this canbe done either as a solution assay, using the techniques outlined aboveor by immobilization on a surface. In the case of proteins, the use ofbeads coated with anti-oculospanin proteins find particular use.

The specimen may be prepared for protein analysis in a variety of waysas will be appreciated by those in the art. In one embodiment, thespecimen is subjected to high-speed centrifugation as necessary toremove insoluble substances, and then prepared as a sample for ELISA/RIAand Western blot.

To prepare a sample for ELISA/RIA, skin or lymph node tissue taken froma subject is used directly, or diluted appropriately in a buffersolution before use. For Western blotting (electrophoresis), a solutionextracted from skin or lymph node tissue can be used directly as thesample, or diluted appropriately with a buffer solution, and mixed witha sample buffer solution (manufactured by Sigma) containing2-mercaptoethanol for SDS-polyacrylamide gel electrophoresis. For dot orslot blotting, a solution extracted from skin or lymph node tissue canbe used undiluted or diluted appropriately in a buffer solution, thesamples are directly adsorbed to a membrane using a blotting device.

A protein in the sample thus obtained can be specifically detected byprecipitating the protein using a procedure such as immunoprecipitationor ligand binding, either without additional immobilization or afterdirect immobilization thereof. For immobilizing a protein, a membraneused can be one such as is used in Western blotting, dot blotting orslot blotting. Examples of such membranes include nitrocellulosemembranes (for example, as manufactured by BioRad), nylon membranes suchas Hybond-ECL (manufactured by Amersham Pharmacia), cotton membranessuch as blot absorbent filters (for example, as manufactured by BioRad)and polyvinylidene difluoride (PVDF) membranes (for example,manufactured by BioRad). IN addition, a variety of protein chips can beused.

To detect and quantify a protein using an ELISA or RIA method, a sampleor a diluted sample solution (for example, diluted with phosphatebuffered saline (hereinafter referred to as “PBS”) containing 0.05%sodium azide) is dispensed into a 96-well plate, such as an Immunoplate,Maxisorp, (manufactured by Nunc) and incubated without agitation at atemperature in the range of 4° C. to room temperature overnight, or at37° C. for 1 to 3 hours, thereby allowing the protein to adsorb thebottom surface of the wells to immobilize the protein.

Antibody against human oculospanin can be obtained using a customarymethod (see, for example, New Biochemical Experimental Course 1, Protein1, p. 389-397, 1992), which comprises immunizing an animal with humanoculospanin or a polypeptide arbitrarily selected from the amino acidsequences of human oculospanin, taking the antibody produced in the bodyand purifying it. Alternatively, a monoclonal antibody can be obtainedin accordance with a method well known in the art (for example, Kohlerand Milstein, Nature 256, 495-497, 1975, Kennet, R. ed., MonoclonalAntibody, p. 365-367, 1980, Prenum Press, N, Y.), which comprises fusingan antibody-producing cell producing an antibody against humanoculospanin with a myeloma cell to form a hybridoma cell.

Human oculospanin protein for use as an antigen can be obtained byintroducing a human oculospanin gene into a host cell by genemanipulation. To explain more specifically, human oculospanin proteinmay be obtained by preparing a vector capable of expressing the humanoculospanin gene, introducing the vector into the host cell, expressingthe gene, and purifying the expressed human oculospanin protein.

The level of expression of human oculospanin can be represented by thelevel of expression of a protein comprising the amino acid sequencerepresented by Sequence ID No. 2 of the sequence listing.

The expression level can be measured by a method known in the art, suchas a Western blotting or a dot/slot blotting method, using anti-humanoculospanin antibody.

Measurement of the level of expression of human oculospanin in aspecimen taken from a healthy person can be performed in the same manneras described above. Then the difference between the level of expressionof the protein measured in the specimen is compared to the level ofexpression in a healthy specimen and thereby detecting that a subjecthas cancer.

The difference in the level of expression of human oculospanin betweenthe specimens from a healthy person and a test subject is analyzed. As aresult, if a specimen exhibits a significantly high expression level ofhuman oculospanin, it can be determined that there is a high probabilityof a subject having cancer, particularly, skin cancer, and moreparticularly, melanoma. In this manner, cancer can be detected.

Alternatively, cancer can be detected by measuring the concentration ofhuman oculospanin and analyzing whether or not the measuredconcentration value falls within the predetermined range for a healthyperson. In this case, if the concentration value of a subject is higherthan the range for a healthy person, it is determined that the subjecthas cancer. Furthermore, by investigating the correlation between thelevel of expression of human oculospanin and the degree of cancerformation in a healthy person, it is possible to determine whether ornot a subject is healthy based on the level of expression of humanoculospanin in a specimen taken from the subject.

5 D Specific Methods for Investigation of the Human Oculospanin Gene andHuman Oculospanin

The human oculospanin gene and human oculospanin are expressed at asignificantly high level in melanocytes in normal human tissues, andthey are, expressed at a significantly higher level in melanoma than innormal melanocytes.

In a method of examining the function of human oculospanin, full-lengthcDNA is first taken from a human cDNA library, derived from cellsexpressing human oculospanin, by a known method such as a colonyhybridization method. Then, the full-length cDNA is introduced into amouse or a human cell, highly-expressed therein, and assessment iscarried out to investigate whether or not the cDNA affects the cell.

To express cDNA in an animal, a method may be used in which thefull-length cDNA obtained is introduced into a virus vector and thevector is administered to the animal. Examples of gene transfectionusing a virus vector include methods of introducing cDNA by integratingit into a DNA virus or an RNA virus, such as a retrovirus, adeno virus,adeno-associated virus, herpes virus, vaccinia virus, pox virus, orpolio virus. Of these, methods using retrovirus, adeno virus,adeno-associated virus and vaccinia virus are preferred.

Examples of non-viral gene transfection include administering anexpression plasmid directly into the muscle (DNA vaccination), liposometreatment, lipofection, micro-injection, calcium phosphate treatment,electroporation and the like. Of these, DNA vaccination and liposometreatment are preferred.

Furthermore, by transfecting full-length cDNA into cultured cells, suchas muscle cells, liver cells, or adipose cells derived from human, mouseor rat; or into primary muscle cells, liver cells, adipose cells or skincells, and expressing the cDNA therein at a high level, it is possibleto examine the functions of a target cell, more specifically, productionand intake of sugars and lipids, control of glycolipid metabolism suchas glycogen accumulation, or to see if there is any effect on themorphology of a cell. Conversely, by introducing into a cell anantisense nucleic acid to the total RNA of a gene to be examined, it ispossible to examine the effects produced on the function and morphologyof the target cell.

To introduce a full-length cDNA into an animal or a cell, the cDNA isintegrated into a vector containing appropriate promoter sequences andtransformation is carried out to transform the host cell with thevector. The expression promoter for use with a vertebrate cell may havea promoter that is typically located upstream of the gene to beexpressed, an RNA splicing site, a polyadenylation site, a transcriptiontermination sequence, etc. Furthermore, if necessary, a replicationinitiation point may be present. Examples of such an expression vectorinclude, but are not limited to, pSV2dhfr having an early promoter ofsimian virus 40 (SV40) (Subramani, S. et al., Mol. Cell. Biol., (1981),1, p854-864), retrovirus vectors pLNCX, pLNSX, pLXIN, pSIR (manufacturedby Clontech), and cosmid vector pAxCw (manufactured by Takara Bio).These expression vectors can be integrated into a simian cell, such as aCOS cell (Gluzman, Y. Cell (1981), 23, p. 175-182, ATCC: CRL-1650), adihydrofolic acid reductase defective strain (Urlaub, G. and Chasin, L.A. Proc. Natl. Acad. Sci. USA (1980), 77, p. 4126-4220) of a Chinesehamster ovary cell (CHO cell, ATCC:CCL-61), human embryonic kidneyderived 293 cell (ATCC: CRL-1573) and the like, by methods including adiethylaminoethyl (DEAE)-dextran method (Luthman, H and Magnusson, G.,Nucleic Acids Res. (1983), 11, p. 1295-1308), a calcium phosphate-DNAco-precipitation method (Graham, F. L. and van der Eb, A. J. Virology(1973), 52, p. 456-457), and an electroporation method (Neumann, E. etal., EMBO J. (1982), 1, p. 841-845). However, the integration method andcell are not limited to those specifically described. In this manner, adesired transformant can be obtained.

Furthermore, using gene manipulation in a healthy animal, a transgenicanimal can be obtained which highly expresses the desired gene. This canbe used to examine the effects on cell phenotype, such as morphology.Alternatively, the state of cells may be examined by preparing aknockout animal by knocking out the target gene in an animal havingmelanoma.

5 E Human Oculospanin Gene and/or Human Oculospanin Detection Kit

The human oculospanin gene and/or human oculospanin can be detectedusing a kit containing one or more components as described herein.Generally, the kit includes nucleic acid primers and/or probes for thedetection of oculospanin. For example, pairs of polymerase chainreaction (PCR) primers for amplifying oculospanin genes can be includedin a kit. These generally comprise oculospanin specific sequences, suchas primers having a continuous sequence of from about 10-15 to about20-30 bases in length for specifically amplifying a polynucleotidecomprising the nucleotide sequence represented by Sequence ID No. 1 ofthe sequence listing. In alternative embodiments, detection probes thathybridize specifically to oculospanin genes are included; in thiscontext, “specificity” means that the oculospanin gene can be identifiedwith little or no cross-hybridization to other genes. In general, thedetection probes have a continuous sequence of at least 10 nucleotidescapable of hybridizing with a polynucleotide comprising the nucleotidesequence represented by Sequence ID No. 1 of the sequence listing understringent conditions, thereby enabling detection of the polynucleotide.Probes can be longer, with about 15, 20 and 25 and upwards nucleotidesall being included. In addition, these detection probes can also belabeled, for example using biotin or fluorophores.

In addition, solid supports can be included in the kits, includingplanar arrays or beads, with immobilized probes in the case of nucleicacid detection, or oculospanin binding proteins such as antibodies, inthe case of protein detection.

In the case of protein detection, oculospanin antigen binding proteins,such as oculospanin antibodies can also be included, and optionally,secondary antibodies capable of binding to an oculospanin antibody.Suitable antibodies are made as described below.

The primer according to section 1) above can be easily constructed basedon the nucleotide sequence of the human oculospanin gene (the nucleotidesequence represented by Sequence ID No. 1 of the sequence listing) by acustomary method, for example, by a method using commercially availableprimer construction software (e.g., Wisconsin GCG package Version 10.2)and subjected to amplification. As an example of such a primer, morespecifically, a primer for amplifying a polynucleotide comprising thenucleotide sequence represented by Sequence ID No. 1 of the sequencelisting, use can be made of the combination of an oligonucleotidecomprising the nucleotide sequence represented by Sequence ID No. 5 ofthe sequence listing and an oligonucleotide comprising the nucleotidesequence represented by Sequence ID No. 6 of the sequence listing. Theprobe according to section 2) above is a polynucleotide capable ofhybridizing specifically with human oculospanin and being 100 to 1500bases in length, preferably 300 to 600 bases in length. These primersand probes may be tagged with an appropriate label (such as an enzymelabel, radioactive label, biotin, or fluorescent label) or may have alinker added thereto.

A kit according to the present invention may contain a thermostable DNApolymerase, dNTPs (a mixture of dATP, dCTP, dGTP and dTTP) and a buffersolution. Examples of thermostable DNA polymerases include Taq DNApolymerase, LA Taq DNA polymerase (manufactured by Takara Shuzo Co.,Ltd.), Tth DNA polymerase, and Pfu DNA polymerase. The type of buffersolution can be selected in accordance with the DNA polymerase which isto be used and Mg²⁺ can be added, as needed.

A kit according to the present invention can be used for detection of ahuman oculospanin gene and/or human oculospanin protein, therebydetermining the presence or absence of cancer and screening for asubstance capable of suppressing cancer growth.

6. Therapeutic Methods

The present invention provides oculospanin binding proteins such asanti-oculospanin antibodies.

6. A. Preparation of Antigen

An antigen for preparing an anti-human oculospanin antibody can be apolypeptide comprising human oculospanin, a partial amino acid sequencethereof having a partial and continuous amino acid sequence comprisingat least 6 bases, or derivatives thereof having an arbitrary amino acidsequence or a carrier added to these (fusion proteins).

Human oculospanin protein can be directly purified from human tumortissues or cells, synthesized in vitro, or produced in host cells bygene manipulation. More specifically, in producing human oculospanin bygene manipulation, a human oculospanin gene is integrated into anexpression vector, and thereafter the human oculospanin is synthesizedin a solution containing enzymes, substrates and energy substancesrequired for its transcription and translation. Alternatively, aprokaryotic or eukaryotic host cell can be transformed with theexpression vector and then human oculospanin can be isolated. Thenucleotide sequence of human oculospanin cDNA is described in: GraemeWistow, Steven L. Bernstein, M. Keith Wyatt, Robert N. Fariss, AmitaBehal, Jeffrey W. Touchman, Gerard Bouffard, Don Smith, and KatherinePeterson (2002), Expressed sequence tag analysis of human RPE/choroidsfor the NEIBank Project: Over 6000 non-redundant transcripts, novelgenes and splice variants, Molecular Vision 8:205-220, and registered inthe GenBank under Accession No. NM_(—)031945. The ORF of the cDNA isshown in Sequence ID No. 1 of the sequence listing. The humanoculospanin cDNA can be obtained from a cDNA library expressing humanoculospanin by using a primer for specifically amplifying humanoculospanin cDNA from the cDNA library as a template through apolymerase chain reaction (hereinafter referred to as the “PCR”, (seeSaiki, R. K., et al., (1988), Science 239, 487-49) herein termed a “PCRmethod”.

The in vitro synthesis for a polypeptide can be performed using, forexample, the rapid translation system (RTS) manufactured by RocheDiagnostics; however, suitable synthesis methods are not limited to thisparticular method. In the case of RTS, the desired gene is cloned intoan expression vector, under the control of a T7 promoter, and theexpression vector is added to an in vitro reaction system. Consequently,mRNA is first transcribed from template DNA by T7 RNA polymerase andthen translation is performed by ribosomes in a solution containingEscherichia coli lysate. In this manner, a target polypeptide can besynthesized in the reaction solution (Biochemica, 1, 20-23 (2001),Biochemica, 2, 28-29 (2001)).

Examples of suitable prokaryotic hosts include Escherichia coli andBacillus subtilis. To transform a desired gene into these host cells,the host cells are transformed with a plasmid vector derived from aspecies compatible with the host, and containing a replicon, that is, areplication initiation point, and a regulatory sequence. Furthermore, itis preferred that the vector has a sequence capable of imparting aselectable phenotype to the cell to be transformed.

As a host cell an Escherichia coli strain, for example, a K12 strain canbe used and pBR322 and pUC series plasmids can generally be used asvectors. However, the choice of host cell and vector is not limitedthereto and any suitable known strain and vector may be used.

Promoters suitable for use in Escherichia coli, include the tryptophan(trp) promoter, lactose (lac) promoter, tryptophan lactose (tac)promoter, lipoprotein (lpp) promoter, and polypeptide chain extensionfactor Tu (tufB) promoter and the like. Any one of these promoters maybe used for producing the desired polypeptide.

As a host cell, a Bacillus subtilis strain can be used, for example, the207-25 strain is preferred. The vector pTUB 228 (Ohmura, K. et al.,(1984), J. Biochem. 95, 87-93) can be used; however, the choice ofBacillus subtilis host and vector is not limited to this particularcombination. By linking a DNA sequence encoding a signal peptidesequence for Bacillus subtilis α-amylase, the protein of interest can beexpressed and secreted from the cell.

Examples of eukaryotic host cells include vertebrate, insect and yeastcells. Examples of vertebrate cells include, but are not limited to, asimian cell, COS cell (Gluzman, Y. (1981), Cell 23, 175-182, (ATCCCRL-1650)), mouse fibroblast cell NIH3T3 (ATCC No. CRL-1658), and adihydrofolic acid reductase defective strain (Urlaub, G. and Chasin, L.A. (1980), Proc., Natl. Acad. Sci, USA 77, 4126-4220) of Chinese hamsterovary cell (CHO cell, (ATCC CCL-61)).

An expression promoter for use with a vertebrate cell, can be one havinga promoter located upstream of the gene to be expressed, an RNA splicingsite, a polyadenylation site, and a transcription termination sequence.Furthermore, a replication initiation site may be present. Examples ofthe suitable expression vectors include, but are not limited to,pCDNA3.1 (manufactured by Invitrogen) having an early promoter of acytomegalo virus and pSV2dhfr (Subramani, S. et al., (1981), Mol. Cell.Biol. 1, 854-864) having an SV40 early promoter.

When using a COS cell or NIH3T3 cell as the host cell, suitableexpression vectors have an SV40 replication initiation site, capable ofself-proliferating in the COS cell or NIH3T3 cell and additionally mayhave a transcription promoter, transcription termination signal, and RNAsplicing site. The expression vector may be integrated into the COS cellor NIH3T3 cell by DEAE-dextran treatment (Luthman, H and Magnusson, G.(1983), Nucleic Acids Res. 11, p. 1295-1308), calcium phosphate-DNAco-precipitation (Graham, F. L. and van der Eb, A. J. (1973), Virology,52, p. 456-457), electroporation (Neumann, E. et al., (1982), EMBO J. 1,p. 841-845) or others. In this manner, a desired transformant cell canbe obtained. Furthermore, when a CHO cell is used as a host cell, if avector capable of expressing a neo gene functioning as an antibioticG418 resistance marker, such as pRSVneo (Sambrook, J. et al., (1989):Molecular Cloning A Laboratory Manual “Cold Spring Harbor Laboratory,NY) or pSV2neo (Southern, P. J., and Berg, P. (1982), J. Mol. Appl.Genet. 1, 327-341) is co-transfected with the expression vector, andthen a G418 resistant colony is selected, a transformed cell stablyproducing the desired polypeptide can be obtained.

The transformant obtained in the manner mentioned above can be culturedin accordance with a customary method to obtain the desired polypeptideexpressed within the cell or secreted outside the cell and thus presentin the culture medium. As a culture medium, various types of mediacustomarily used can be selected appropriately depending upon the typeof host cell employed. More specifically, for COS cells, RPMI 1640medium or Dulbecco's Modified Eagle's medium (hereinafter referred to as“DMEM”) may be used. If necessary, serum components such as fetal calfserum may be added to the medium.

A recombinant protein produced within a cell or secreted outside atransformant cell and present in the culture medium can be separated andpurified by various known separation methods on the basis of thephysical properties and chemical properties of the protein. Examples ofsuch separation methods include treatment with a general proteinprecipitating agent, ultrafiltration, molecular sieve chromatography(gel filtration), adsorption chromatography, ion-exchangechromatography, affinity chromatography, various types of liquidchromatographic methods such as high-performance liquid chromatography(HPLC), dialysis and combinations of these methods. If a hexa-his tag isfused to the recombination protein which is expressed, the recombinantprotein can be efficiently purified by a nickel affinity column. If theaforementioned methods are used in combination, a large amount of adesired polypeptide can be obtained with a high purity and in a highyield.

Alternatively, the antigen used can be a membrane fraction prepared froma recombinant cell expressing human oculospanin or a recombinant cellexpressing human oculospanin, or a chemically synthesized peptidefragment of a protein according to the present invention obtained by amethod known to those skilled in the art.

Once the antigen is made, antibodies can be produced.

6. B. Production of Anti-Human Oculospanin Monoclonal Antibody

An example of an antibody which specifically binds to human oculospanin,is a monoclonal antibody which specifically binds to human oculospanin.A method suitable for obtaining such monoclonal antibody is as follows:

To produce the monoclonal antibody, the steps necessary requiredinclude:

(a) purifying the biomacromolecule which is to be used as an antigen;

(b) immunizing an animal by injecting the antigen into the animal,taking a blood sample and checking the antibody titer to determine thetime at which the spleen should be excised, and preparing antibodyproducing cells;

(c) preparing bone myeloma cells (hereinafter referred to as “myeloma”);

(d) fusing the antibody-producing cells and the myeloma;

(e) selecting hybridomas producing a desired antibody;

(f) segregating (cloning) them into single cell clones;

(g) optionally, culturing the hybridoma to produce a large amount ofmonoclonal antibody or raising an animal having the hybridomatransplanted therein; and

(h) analyzing the physiological activity and binding specificity of themonoclonal antibody thus produced, or characteristics of the monoclonalantibody as a labeling agent.

The method of producing a monoclonal antibody is described in moredetail below in accordance with the steps mentioned above. However,methods of producing monoclonal antibody are not limited to the methoddescribed. For example, an antibody-producing cell other than a spleencell and myeloma.

6. C Preparation of an Antibody Producing Cell

An antigen obtained as above is mixed with Freund's complete orincomplete adjuvant or an auxiliary agent such as potassium aluminumsulfate. The mixture is used as an immunogen and is injected into ananimal. A suitable experimental animal would be an animal known to besuitable for use in a hybridoma preparation method. Specific examples ofsuch animals include mice, rats, goats, sheep, cows and horses. However,in view of the availability of myeloma cells which are to be fused withthe antibody-producing cells taken from the animal, mice or rats arepreferred as the animals to be immunized. The choice of strains of miceor rats used in practice is not particularly limited. Examples ofsuitable mouse strains include A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL,C57BR, C57L, DBA, FL HTH, HTI, LP, NZB, NZW, RF, R III, SJL, SWR, WB,and 129. Examples of rat strains include Low, Lewis, Spraque, Daweley,ACI, BN, and Fischer. These mice and rats are available fromexperimental animal-raising distributors such as Clea Japan Inc.,Charles River Japan Inc., Japan SLC Inc., and The Jackson Laboratories.In view of fusion compatibility with myeloma cells as discussed later,“BALB/c” as a mouse line and “Low” as a rat line are particularlypreferred as the immunized animal. In consideration of homology of anantigen between a human and a mouse, a mouse having a reduced biologicalfunction for removing autoantibody, in other words, a mouse sufferingfrom autoimmune disease is preferably used. Note that a mouse or a ratwhich is to be immunized is preferably 5 to 12 weeks old, morepreferably 6 to 8 weeks old.

An animal can be immunized with human oculospanin or a recombinantlyproduced version thereof by known methods, such as the methodsspecifically described in, for example, Weir, D. M. Handbook ofExperimental Immunology Vol. I. II. III., Blackwell ScientificPublications, Oxford (1987), Kabat, E. A. and Mayer, M. M., ExperimentalImmunochemistry, Charles C Thomas Publisher Springfield Ill. (1964),etc. Of these immunization methods, a method preferably used in thepresent invention is, for example, performed as follows. First, anantigen, that is, a membrane protein fraction, or a cell expressing anantigen, is injected into an animal intradermally or intraperitoneally.To improve immunization efficiency, both injection methods can be usedtogether. More specifically, when the intradermal injection is performedin the first half of the injections and the intraperitoneal injection isperformed in the second half of the injections or only the last time,the immunization efficiency can be particularly increased. The dosingregimen of the antigen differs depending upon the type and individualdifferences, etc. of the animal body to be immunized. However, theantigen is preferably injected 3 to 6 times at intervals of 2 to 6weeks, and more preferably 3 to 4 at intervals of 2 to 4 weeks. It ispreferred not to excessively increase the number of dosings, becausethen the antigen may be wasted. Also, it is preferred not to overlyextend the length of the dosing interval, because the activity of thecells decreases due to aging of the animal. The dose of the antigendiffers depending upon the type and individual differences, etc. of theanimal body; however, the dose generally falls within the range of about0.05 to 5 ml, preferably about 0.1 to 0.5 ml. Booster immunization isperformed 1 to 6 weeks after the antigen is administered, preferablyafter 2 to 4 weeks, more preferably after 2 to 3 weeks. If the boosterimmunization is performed after more than 6th weeks or within 1 week,the booster immunization will be less effective. Note that the dose ofthe antigen to be injected as a booster differs depending upon the typeand size of the animal body; however, for example, for mice, itgenerally falls within the range of about 0.05 to 5 ml, preferably about0.1 to 0.5 ml, and more preferably about 0.1 to 0.2 ml. It is preferablenot to administer an unnecessarily large amount of antigen because thenthe immunization effect decreases and it is unfavorable to the animal tobe immunized.

One to 10 days, preferably, 2 to 5 days, more preferably 2 to 3 daysafter the booster immunization, spleen cells or lymphocytes containingantibody-producing cells are removed from the immunized animal underaseptic conditions. At this time, an antibody titer is determined. If ananimal having a sufficiently high antibody titer is used as the supplysource for the antibody-producing cells, the efficiency of the followingoperations can be enhanced. As a method of determining the antibodytiter to be used herein, various types of known technologies areappropriate, such as RIA methods, ELISA methods, fluorescent antibodymethods, and passive blood cell agglutination reaction methods. In viewof detection sensitivity, speed, accuracy, and the possibility ofautomatic operation, RIA methods and ELISA methods are preferred.

The determination of an antibody titer according to the presentinvention can be performed by an ELISA method as follows. First, thepurified or partially purified antigen is adsorbed onto a solid surfacesuch as 96-well plate for ELISA. Then, solid surface having no antigenadsorbed thereon is covered with a protein unrelated to the antigen,such as bovine serum albumin (hereinafter referred to as “BSA”). Afterwashing the surface, the surface is brought into contact with aserially-diluted sample (e.g., mouse serum) serving as a primaryantibody, thereby allowing a monoclonal antibody contained in the sampleto bind to the antigen. Furthermore, a secondary antibody, that is, anenzyme-labeled antibody against a mouse antibody, is added to bind tothe mouse antibody. After washing the resultant complex, a substrate forthe enzyme is added and the change in absorbance, which occurs due to acolour change induced by degradation of the substrate, is measured tocalculate the antibody titer.

Antibody-producing cells are separated from the spleen cells orlymphocytes in accordance with known methods (for example, described inKohler et al., Nature, 256, 495, 1975; Kohler et al., Eur J. Immunol.,6, 511, 1977; Milstein et al., Nature, 266, 550, 1977; Walsh, Nature,266, 495, 1977). More specifically, in the case of spleen cells, theantibody-producing cells can be separated by a general method whichcomprises homogenizing tissue, filtering the homogenized through astainless steel mesh, and suspending the cells obtained in Eagle'sMinimum Essential Medium (MEM).

6. C i) Preparation of Bone Myeloma Cells (Hereinafter Referred to as“Myeloma”)

The choice of myeloma cells which are to be used for cell fusion is notparticularly limited and suitable cells can be selected from known cellstrains. For convenience when hybridoma are selected from fused cells,it is preferable to use a HGPRT (Hypoxanthine-guanine phosphoribosyltransferase) defective strain whose selection procedure has beenestablished. More specifically, examples of HGPRT defective strainsinclude X63-Ag8(X63), NSI-Ag4/1(NS1), P3×63-Ag8.U1(P3U1),X63-Ag8.653(X63.653), P2/0-Ag14(SP2/0), MPC11-45.6TG1.7(45.6TG), F0,S149/5XXO and BU.1 derived from mice, 210.RSY3.Ag.1.2.3 (Y3) derivedfrom rat; and U266AR(SKO-007), GM1500.GTG-A12(GM1500), UC729-6,LICR-LOW-HMy2(HMy2), and 8226AR/NIP4-1(NP41) derived from humans. TheseHGPRT defective strains are available from the American Type CultureCollection (ATCC), etc.

These strains are subcultured in an appropriate medium such as8-azaguanine medium [RPMI-1640 supplemented with glutamine,2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafterreferred to as “FCS”) and further 8-azaguanine is added thereto];Iscove's Modified Dulbecco's Medium (hereinafter referred to as “IMDM”),or Dulbecco's Modified Eagle Medium (hereinafter referred to as “DMEM”).In this case, 3 to 4 days before performing the cell fusion operation,the cells are transferred to a regular medium [for example, ASF104medium (manufactured by Ajinomoto Co. Inc.) containing 10% FCS] andsubcultured therein to obtain not less than 2×10⁷ cells by the day ofcell fusion.

6 C ii) Cell Fusion

Fusion between antibody-producing cells and myeloma cells isappropriately performed in accordance with known methods (including:Weir, D. M. Handbook of Experimental Immunology Vol. I. I. III.,Blackwell Scientific Publications, Oxford (1987), Kabat, E. A., andMayer, M. M. Experimental Immunochemistry, Charles C Thomas Publisher,Springfield, Ill. (1964)), under conditions such that the survival rateof cells is not excessively reduced. Examples of such methods includechemical methods in which antibody-producing cells and myeloma cells aremixed in a high concentration polymer solution, for example,polyethylene glycol; and physical methods using electric stimulation. Ofthese methods, the chemical method is more specifically explained asfollows. When polyethylene glycol is used as the high concentrationpolymer solution, antibody-producing cells and myeloma cells are mixedin a solution of polyethylene glycol having a molecular weight of 1,500to 6,000, more preferably, 2,000 to 4,000, at a temperature of 30 to 40°C., preferably 35 to 38° C., for 1 to 10 minutes, more preferably 5 to 8minutes.

6. C. iii) Selection of Hybridoma Populations

The method of selecting hybridoma obtained by cell fusion is notparticularly restricted. Usually, use is made of the HAT (hypoxanthine,aminopterin, thymidine) selection method [Kohler et al., Nature, 256,495 (1975); Milstein at al., Nature 266, 550 (1977)]. This is aneffective method when hybridoma are obtained using myeloma cells of aHGPRT defective strain incapable of surviving in the presence ofaminopterin. More specifically, by culturing unfused cells and hybridomain HAT medium, only hybridoma having aminopterin resistance are selectedand allowed to remain and proliferate.

6. C iv) Segregation to Single Cell Clone (Cloning)

As a cloning method for hybridoma, known methods such as amethylcellulose method, soft agarose method, or limiting dilution methodcan be used [see, for example, Barbara, B. M. and Stanley, M. S.:Selected Methods in Cellular Immunology, W.H. Freeman and Company, SanFrancisco (1980)]. Examples of a cloning method include a limitingdilution method in which hybridoma cells are diluted so as to contain asingle hybridoma cell per well of a plate and cultured; a soft agarosemethod in which hybridoma cells are cultured in a soft agarose mediumand colonies are recovered; a method of taking individual hybridomacells by means of a micro manipulator and culturing them; and aso-called “clone sorter method” in which hybridoma cells are separatedone by one by means of a cell sorter. Of these methods, the limitingdilution method is preferred. In this method, a fibroblast cell strainderived from a rat fetus or feeder cells such as healthy mouse spleencells, thymus gland cells, or ascites cells are seeded. Hybridoma cellsare diluted in medium to provide a dilution ratio of 0.2 to 0.5 cellsper 0.2 ml. The diluted hybridoma suspension solution is transferredinto wells to provide 0.1 ml per well and continuously cultured forabout 2 weeks with changes of about ⅓ of the medium with fresh medium atpredetermined time intervals (for example, every 3 days). In thismanner, hybridoma clones can be proliferated.

The hybridoma cells in the well for which antibody titer has beenconfirmed are subjected to repeat cloning by the limiting dilutionmethod, 2 to 4 times. Hybridoma cells, with an antibody titer which isconfirmed to be stable, are selected as anti-human oculospaninmonoclonal antibody producing hybridoma strains. One of the clonedhybridoma strains thus obtained is designated as “O3B8-2C9-4F3” and thishas been deposited at the International Patent Organism Depositary ofthe National Institute of Advanced Industrial Science Technology(located at Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan)as of Feb. 17, 2004 under deposition No. FERM BP-08627.

6 C v) Preparation of Monoclonal Antibody by Culturing Hybridoma Cells

The hybridoma cells thus selected are cultured to efficiently obtainmonoclonal antibody. However, prior to culturing, it is desirable that ahybridoma cell producing a desired monoclonal antibody is screened. Thescreening is performed by a known method.

The determination of antibody titer can be performed in the presentinvention by, for example, an ELISA method in accordance with thefollowing procedure. First, purified or partially purified humanoculospanin or cells expressing human oculospanin are adsorbed onto asolid surface of a 96-well plate for ELISA. Then, the solid surfacehaving no antigen adsorbed thereon is covered with a protein unrelatedto the antigen, for example, bovine serum albumin (hereinafter referredto as “BSA”). After washing the surface, the surface is brought intocontact with a serially-diluted sample (for example, mouse serum) as afirst antibody, thereby allowing binding of an anti-human oculospaninantibody in the sample to the antigen. Furthermore, an antibody againstthe mouse antibody and labeled with an enzyme, serving as a secondaryantibody, is added to bind to the mouse antibody. After washing theresultant complex, a substrate for the enzyme is added and the change ofabsorbance, which occurs due to the colour change induced by degradationof the substrate, is determined to calculate the antibody titer. In thisway, the antibody titer is calculated. Note that such a screeningoperation can be performed after or before cloning of the hybridoma cellas mentioned above.

A hybridoma obtained by the aforementioned method can be stored in afrozen state in liquid nitrogen or in a refrigerator at 80° C. or less.

After completion of cloning, hybridoma are transferred from HT medium toa general medium and cultured. Large-scale culture is performed byrotation culture using a large culture bottle or by spinner culture. Thesupernatant obtained from the large-scale culture is purified by a knownmethod to those skilled in the art, such as gel filtration, to obtain amonoclonal antibody which specifically binds to a protein according tothe present invention. The hybridoma can be injected into the abdominalcavity of a mouse of the same line as the hybridoma (for example,BALB/c) or a Nu/Nu mouse to proliferate the hybridoma. In this way,ascites fluid containing a large amount of the monoclonal antibodyaccording to the present invention can be obtained. When hybridoma cellsare injected into the abdominal cavity, if a mineral oil such as2,6,10,14-tetramethyl pentadecane (pristine) has (3 to 7 days before)been administered previously, the ascites fluid can be obtained in alarger amount. To explain more specifically, an immunosuppressive agentis previously injected into the abdominal cavity of a mouse of the samestrain as the hybridoma. Twenty days after inactivation of the T cells,10⁶ to 10⁷ of hybridoma clone cells are suspended in a serum-free medium(0.5 ml) and the suspension is injected into the abdominal cavity. Whenthe abdomen is expanded and filled with the ascites fluid, the ascitesfluid is taken. By virtue of this method, the monoclonal antibody can beobtained at a concentration 100-fold higher than that of the culturemedium.

A monoclonal antibody obtained in the aforementioned method can bepurified by the methods described in, for example, Weir, D. M.: Handbookof Experimental Immunology Vol. I, II, III, Blackwell ScientificPublications, Oxford (1978). To explain more specifically, examples ofsuch methods include ammonium sulfate precipitation methods,gel-filtration methods, ion exchange chromatographic methods, andaffinity chromatographic methods. Of these, the ammonium sulfateprecipitation method, if it is repeated 3 to 4 times, preferably 3 to 6times, successfully purifies the monoclonal antibody. However, in thismethod, the yield of the purified monoclonal antibody is extremely low.Therefore, the monoclonal antibody is crudely purified by performing theammonium sulfate precipitation method once or twice and then subjectedto at least one method, and preferably two methods, selected from gelfiltration, ion exchange chromatography, and affinity chromatography andthe like. In this way, highly purified monoclonal antibody can beobtained in a high yield. The ammonium sulfate precipitation method maybe performed in the following combination and in the following order: a)ammonium sulfate precipitation method—ion exchange chromatographicmethod-gel filtration method; b) ammonium sulfate precipitationmethod—ion exchange chromatographic method—affinity chromatographicmethod; and c) ammonium sulfate precipitation method—gel filtrationmethod—affinity chromatographic method, etc. Of these combinations, toobtain the monoclonal antibody with a high purity in a high yield,combination c) is particularly preferable.

As a simple purification method, a commercially available antibodypurification kit (for example, MAbTrap GII kit manufactured byPharmacia) and the like can be used.

The monoclonal antibody thus obtained has high antigen specificity forhuman oculospanin.

6 C vi) Analysis of Monoclonal Antibody

The monoclonal antibody thus obtained is checked for isotype andsubclass thereof as follows. Suitable identification methods include theOuchterlony method, ELISA methods and RIA methods. The Ouchterlonymethod is simple; although, if monoclonal antibody is obtained at lowconcentration it must be concentrated. Alternatively, when an ELISAmethod or RIA method is used, the culture supernatant can be directlyreacted with an antigen adsorption solid phase. In addition, if varioustypes of antibodies corresponding to immunoglobulin isotypes andsubclasses are used as secondary antibodies, the isotype and subclass ofthe monoclonal antibody can be identified. As a further simple method, acommercially available identification kit (for example, Mouse Typer kitmanufactured by BioRad) and the like can be used.

The quantification of a protein can be performed by the Folin Lowryassay based on the adsorption at 280 nm [1.4 (OD280)=Immunoglobulin 1mg/ml].

6 C vii) Method Using a Polymerase Chain Reaction

When the amino acid sequence of a desired protein has been elucidated inits entirety or in part, oligonucleotide primers of a sense strand andan antisense strand corresponding to a part of the amino acid sequenceare synthesized. Then, the polymerase chain reaction [Saiki, R. K., etal. (1988) Science 239, 487-49] is performed by using these primers incombination to amplify a DNA fragment encoding heavy chain and lightchain subunits of a desired anti-human oculospanin antibody. As thetemplate DNA used herein, use may be made of cDNA synthesized from mRNAof a hybridoma producing the anti-human oculospanin monoclonal antibodyby a reverse transcriptase reaction.

The DNA fragment thus prepared can be directly integrated into a plasmidvector by use of a commercially available kit, etc. Alternatively, theDNA fragment may be used for selecting a desired clone by labeling thefragment with ³²P, ³⁵S, or biotin, and performing colony hybridizationor plaque hybridization by using it as a probe.

For example, a method of examining a partial amino acid sequence of eachsubunit of the anti-human oculospanin monoclonal antibody of the presentinvention is preferably performed by isolating each subunit by use of aknown method such as electrophoresis or column chromatography and thenanalyzing the N-terminal amino acid sequence of each subunit using anautomatic protein sequencer (for example, PPSQ-10, manufactured byShimadzu Corporation).

A method of isolating cDNA encoding each subunit of the anti-humanoculospanin monoclonal antibody protein from the desired transformantobtained as mentioned above is performed in accordance with a knownmethod [see Maniatis, T., et al. (1982) in “Molecular Cloning ALaboratory Manual” Cold Spring Harbor Laboratory, NY.], and morespecifically, can be performed by separating fractions corresponding tovector DNA from a cell and excising a DNA region encoding a desiredsubunit from the vector DNA (plasmid DNA).

(b) Screening Method Using a Synthesized Oligonucleotide Probe **TT

When the whole or part of the amino acid sequence of a desired proteinis elucidated (any sequence is taken from any region of the desiredprotein as long as it is a specific sequence having a plurality ofcontiguous amino acids), an oligonucleotide is synthesized (in thiscase, use may be made of either a nucleotide sequence presumed based onthe degree of frequency of codons in use or a plurality of nucleotidesequences of conceivable nucleotide sequences in combination; in thelatter case, the number of types of nucleotide sequences can be reducedby integrating inosine) so as to correspond to the amino acid sequence,used as a probe (labeled with ³²P, ³⁵S or biotin); hybridized with anitrocellulose filter on which the DNA of a transformant strain isdenatured and immobilized, and then the positive strain obtained isisolated.

The sequence of the DNA thus obtained can be determined by theMaxam-Gilbert chemical modification method [see Maxam, A. M. andGilbert, W. (1980) in “Methods in Enzymology” 65, 499-576] and thedideoxynucleotide chain termination method [Messing, J. and Vieira, J.(1982) Gene 19, 269-276].

Recently, an automatic base sequence determination system using afluorescent dye has been widely used (for example, sequence robots“CATALYST 800” and model 373ADNA sequencer, etc. manufactured byPerkinElmer Japan Co., Ltd.)

Using such a system also makes it possible to efficiently and safelydetermine a DNA nucleotide sequence. Based on the data of the presentinvention thus determined including the nucleotide sequence of DNA andthe N-terminal amino acid sequences of the heavy chain and light chain,it is possible to determine the entire amino acid sequence of the heavychain and light chain of the monoclonal antibody of the presentinvention.

The heavy chain and light chain of immunoglobulin each constitute avariable region and a constant region. The variable region furtherconstitutes complementarity-determining regions (hereinafter referred toas “CDR”, there are 3 sites in each of the heavy chain and light chain)and framework regions adjacent to these CDRs (4 sites in each of theheavy chain and light chain).

The amino acid sequence of the constant region is common to antibodiesbelonging to the same immunoglobulin class regardless of the type ofantigen. In the variable region, the amino acid sequence of a CDR isintrinsic to each antibody. However, according to a study comparingamino acid sequence data of numerous antibodies, it is known that theposition of the CDR and the length of a framework sequence are similarbetween the subunits of different antibodies as long as they belong tothe same subgroup [see Kabat, E. A., et al. (1991) in “Sequence ofProteins of Immunological Interest Vol. II”: U.S. Department of Healthand Human Services]. Therefore, it is possible to determine the positionof the CDRs and framework regions and further the constant region ineach amino acid sequence, by comparing the amino acid sequences of theheavy chain and the light chain of the anti-human oculospanin monoclonalantibody of the present invention with the known amino acid sequencedata. Note that the chain length of FRH₁, that is, the framework regionlocated at the side proximal to the N terminus, is sometimes shorterthan the general length of 30 amino acids. In some cases, the frameworkregion is known to have a minimum of 18 amino acids [see Kabat et al.cited above]. From this, in the antibody of the present invention, thechain length of the framework region at the N-terminus of the heavychain is set at 18 to 30 amino acids, preferably 30 amino acids, as longas the function of the anti human oculospanin antibody is not impaired.

In summary, only by artificially modifying a peptide having the sameamino acid sequence as each of the CDRs of light chains or heavy chainsor a partial contiguous amino acid sequence thereof, as determinedabove, thereby approximating the structure to the tertiary structure ofthe CDR actually taken from within the anti-human oculospanin antibodymolecule, a binding activity capable of binding to human oculospanin canbe imparted to the CDR [see, for example, U.S. Pat. No. 5,331,573].Hence, a peptide containing the same amino acid sequence as that of aCDR or a partial amino acid sequence thereof is also included as being amolecule of the present invention.

A modified amino acid sequence can be prepared by deleting at least oneor more amino acids from its original amino acid sequence in accordancewith cassette mutagenesis [see Toshimitu Kishimoto, “New BiochemicalExperimental Lecture 2, Nucleic acid III, Recombinant DNA technique”, p242-251].

Such various types of DNA sequences can be produced in accordance with acustomary method for chemically synthesizing a nucleic acid, forexample, the phosphite triester method [see Hunkapiller, M., et al.(1984) Nature 310, 105-111]. Note that codons corresponding to a desiredamino acid are already known per se. Any codon may be selected.Alternatively, which codon is used can be determined in accordance witha customary method by considering the frequency with which codons areused by the host cell. The partial modification of the nucleotidesequences of codons, may be performed in accordance with a customarymethod, more specifically, in accordance with a site-specificmutagenesis method [see Mark, D. F., et al. (1984) Proc. Natl. Acad.Sci. USA 81, 5662-5666] using a synthetic oligonucleotide primerencoding a desired modification.

Furthermore, it is possible to check whether a certain type of DNA canhybridize with DNA encoding a heavy chain or light chain of ananti-human oculospanin monoclonal antibody of the present invention bysubjecting the DNA to the following experiment performed using a probeDNA labeled with [α-³²P]dCTP, in accordance with the random primermethod [see Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem.132, 6-13] or the nick translation method [see Maniatis, T., et al.(1982) in “Molecular Cloning A laboratory Manual” Cold Spring HarborLaboratory, NY.].

To explain more specifically, the DNA to be checked is adsorbed onto,for example, a nitrocellulose or nylon membrane. After it is denaturedwith alkali, if necessary, the membrane is heated or UV-irradiated,thereby immobilizing the DNA onto the membrane. The membrane is soakedin a pre-hybridization solution containing 6×SSC (1×SSC contains 0.15Msodium chloride, 0.015 trisodium citrate solution) and 5% Denhardt'ssolution, and 0.1% sodium dodecylsulfate (SDS), and maintained at 55° C.for 4 hours or more. Subsequently, the probe prepared in advance isadded to the pre-hybridization solution so as to have a final specificactivity of 1×10⁶ cpm/ml and the temperature is maintained at 60° C.overnight. Thereafter, the membrane is washed with 6×SSC at roomtemperature for 5 minutes several times, further washed with 2×SSC for20 minutes and subjected to autoradiography.

Using the aforementioned methods, DNA which hybridizes with the DNAencoding a heavy chain or light chain of the humanized anti-humanoculospanin antibody of the present invention can be isolated from arandom cDNA library or a genomic library [see Maniatis, T., et al.(1982) in “Molecular Cloning A Laboratory Manual” Cold Spring HarborLaboratory, NY.].

Each of the DNA sequences obtained in the aforementioned manner can beintegrated into an expression vector, which can be then introduced intoa prokaryotic or eukaryotic host cell. In this way, the gene (having theDNA) can be expressed in the host cell as described herein.

A fraction containing an anti-human oculospanin antibody proteinproduced within or outside the transformant cell can be treated byvarious known protein isolation procedures based on the use of physicaland/or chemical properties to isolate and purify the protein. Examplesof these methods include treatment with a protein precipitation agentgenerally used, ultrafiltration, chromatography, such as molecular sievechromatography (gel filtration), adsorption chromatography, ion-exchangechromatography, and affinity chromatography, or high performance liquidchromatography (HPLC), dialysis, and combinations thereof.

To humanize the anti-human oculospanin monoclonal antibody, the aminoacid sequence of a variable region must be designed such that the entireCDR sequence and a partial amino acid sequence of the FR sequencedetermined are transplanted into a human antibody framework, as follows:

Conventionally, in designing a humanized antibody, an acceptor subgroupis selected based on the following guidelines.

a) the natural combination of a heavy chain and light chain of a knownhuman antibody having a naturally occurring amino acid sequence is usedas it is;

b) although the combination of a heavy chain and a light chain as asubgroup is maintained; the heavy chain and the light chain may bederived from different human antibodies. The heavy chain and the lightchain which are to be used may be selected from amino acid sequenceswith high identity to those of the heavy chain and light chain of thedonor, respectively, and the consensus sequences. In the presentinvention, the aforementioned guidelines may be employed. However, thereare alternative methods as follows:

c) regardless of consideration of the combination of the subgroup, amethod may be employed for selecting FRs of the heavy chain and lightchain with high identity to those of a donor from the library of primarysequences of a human antibody. In these selection methods, the degree ofidentity of the amino acids of the FR region between a donor and anacceptor can be set at 70% or more. By employing such a method, it ispossible to reduce the number of amino acid residues of an antibody tobe transplanted from a donor, thereby inducing less HAMA response.

There is an operation (hereinafter referred to as “molecular modeling”)for predicting the tertiary structure of an antibody molecule from itsprimary sequence; however, the accuracy of prediction of this operationis limited. Therefore, the role of an amino acid residue appearing onlyrarely in the subgroup to which the donor belongs cannot be sufficientlyspecified. It is generally difficult to determine which amino acidresidue of a donor or an acceptor should be selected for such a positionof the amino acid residue in accordance with the method described aboveby Queen and co-workers. However, in accordance with the selectionmethod (c), it is possible to reduce the frequency with which suchdetermination must be made.

The present inventors have further improved such humanization methods byproviding a novel method of identifying an amino acid derived from theFR of a donor and important for maintaining the structure and functionof a CDR of the donor.

After a human acceptor molecule for each of a light chain and heavychain is selected, the amino acid residue to be transferred from the FRof a donor is selected by the method mentioned below.

In the amino acid sequences of the donor and the acceptor, when thecorresponding amino acid residues of their FRs differ from each other,it must be determined which amino acid residue should be selected. Whenmaking such a selection, care must be taken so as not to damage thetertiary structure of the CDR derived from the donor.

Queen et al. have proposed, in the Japanese National Publication ofInternational Patent Application No. 4-502408, a method of transplantingan amino acid residue on the FR into an acceptor together with a CDRsequence, if it satisfies at least one of the following conditions.

1) The amino acid is rarely present at the position within a human FRregion of an acceptor, whereas the corresponding amino acid of a donoris usually present at the equivalent position;

2) the amino acid is located extremely close to one of the CDRs;

3) it is predicted that the amino acid has a side chain atom withinabout 3 angstroms from the CDR in its three dimensional immunoglobulinmodel and the side chain atom can interact with an antigen or the CDR ofa humanized antibody.

In the above, a residue satisfying requirement 2) above often exhibitsthe property of requirement 3). Therefore, in the present invention,requirement 2) is omitted and two requirements are newly set. Morespecifically, in the present invention, if the amino acid residue on thedonor's FR to be transferred together with the CDR satisfies thefollowing:

a) the amino acid is rarely present at the position within an FR regionof an acceptor, whereas the corresponding amino acid of a donor isusually present at the equivalent position;

b) in the tertiary structure model, the amino acid presumably interactswith a constituent amino acid atom of the CDR and an antigen or the CDRloop to be transplanted;

c) the position mentioned above is that of a canonical classdetermination residue; or

d) the position is that which forms a contact surface between a heavychain and a light chain,

then the amino acid residue is transplanted from the FR of the donor.

In requirement a), in accordance with the Kabat list mentioned above, anamino acid found at a frequency of 90% or more at a position in the samesubclass of antibody is defined as “usually present”, whereas an aminoacid found at a frequency of less than 10% is defined as “rarelypresent”.

In requirement c), as to whether or not “the position mentioned above isa canonical class determining residue”, the determination can be madeuniquely in accordance with Chothia's list as mentioned above.

In requirements b) and d), molecular modeling of the antibody's variableregion must be performed in advance. As software for molecular modeling,any commercially available software may be used; however, preferably AbM(manufactured by Oxford Molecular Limited Company) is used.

The accuracy of prediction by molecular modeling is somewhat limited.Therefore, in the present invention, by considering X-raycrystallographic data for variable regions of various antibodies, thereliability of the structure predicted by molecular modeling can beevaluated in two steps.

In the tertiary structure of the variable region constructed by themolecular modeling software, such as AbM, if the distance between twoatoms is shorter than a value of the sum of the van der Waals radius oftwo atoms plus 0.5 angstroms, the two molecules are assumed to be in vander Waals contact. On the other hand, if the distance between atomshaving polarity, such as amide nitrogen or carbonyl oxygen, of the mainand side chains, is shorter than a distance of an average hydrogenbinding distance, 2.9 angstroms plus 0.5 angstroms, it is assumed thathydrogen bonding may exist between the atoms. Furthermore, if thedistance between the oppositely charged atoms is shorter than a distanceof 2.85 angstroms plus 0.5 angstroms, it is assumed that an ionic bondis formed between the atoms.

On the other hand, from X-ray crystallographic experimental results forvariable regions of various antibodies, as the position on the FR atwhich contact with the CDR can be found with a high frequency regardlessof the subgroup, the following positions can be specified: in the lightchain, the 1, 2, 3, 4, 5, 23, 35, 36, 46, 48, 49, 58, 69, 71, and 88thpositions, and in the heavy chain, 2, 4, 27, 28, 29, 30, 36, 38, 46, 47,48, 49, 66, 67, 69, 71, 73, 78, 92, 93, 94, and 103rd positions(numerals all represent amino acid numbers defined in the documentsdescribed by Kabat et al. The same definition will be also appliedbelow). When the same standard as that of the molecular modeling isapplied, the amino acid residues of these positions are confirmed to bein contact with the amino acid residues of the CDR in the 2/3 portion ofthe known antibody's variable region. Based on the findings, thesentence: “b) In the tertiary structure model, the amino acid presumablyinteracts with a constituent amino acid atom of the CDR and an antigenor the CDR loop to be transplanted” means as follows.

In molecular modeling, if a position in the FR which is expected to bein contact with the CDR agrees with any one of the positions at whichthe contact between the FR and the CDR is reported to frequently occuraccording to experimental detection by X-ray crystallography, selectionof the amino acid residue from the donor is preferred. In other cases,requirement b) is not taken into consideration.

The sentence: “d) the position is that which forms a contact surfacebetween the heavy chain and the light chain” means the followingrequirement. From the X-ray crystallographic experimental results forthe variable regions of various antibodies, it is confirmed that heavychain-light chain contact is frequently observed at the 36, 38, 43, 44,46, 49, 87, 98th amino acid residues in the light chain and at the 37,39, 45, 47, 91, 103, and 104th amino acid residues in the heavy chain.In cases where the possibility of heavy chain-light chain contact ispredicted in the molecule modeling and the contact position agrees withany one of the aforementioned positions, transplantation of the aminoacid residue from the donor is preferably performed. In other cases,requirement d) is not taken into consideration.

The DNA encoding variable regions of the heavy chain and light chain ofa humanized anti-human oculospanin antibody of the present invention canbe produced by the methods described below.

For example, a plurality of polynucleotide fragments comprising apartial nucleotide sequence of the DNA, of 60 to 70 nucleotides inlength, are chemically synthesized alternately from the sense andantisense strands. Thereafter, individual polynucleotide fragments areannealed and ligated using DNA ligase. In this way, it is possible toobtain a DNA having DNA encoding variable regions of the heavy chain andlight chain of a desired humanized anti-human oculospanin antibody.

In another method, DNA encoding the total amino acid sequence of thevariable region of an acceptor is extracted from human lymphocytes,replacement of nucleotides is performed in the region encoding a CDR bya method known to those skilled in the art to introduce a restrictionenzyme cleavage sequence. After the region is cleaved with thecorresponding restriction enzyme, the nucleotide sequence encoding a CDRof the donor is synthesized and ligated using DNA ligase. In this way,it is possible to obtain the DNA encoding variable regions of the heavychain and light chain of a desired humanized anti-human oculospaninantibody.

Furthermore, in the present invention, it is possible to obtain DNAcomprising DNA encoding variable regions of the heavy chain and lightchain of a desired humanized anti-human oculospanin antibody, preferablyin accordance with the overlap extension PCR method (Horton et al.,Gene, 77, 61-68, (1989)) described below.

To explain more specifically, two different DNA sequences, which encodetwo different amino acid sequences, respectively and which are desiredto be ligated to each other, are designated as (A) and (B), for the sakeof convenience. A sense primer of 20 to 40 nucleotides (hereinafterreferred to as a “primer (C)”) to be annealed to the 5′ side of the DNAsequence (A) and an antisense primer of 20 to 40 nucleotides(hereinafter referred to as a “primer (D)) to be annealed to the 3′ sideof the DNA sequence (B) are chemically synthesized. Furthermore, achimeric-type sense primer (hereinafter referred to as “primer (E)) isformed by ligating a nucleotide sequence of 20 to 30 nucleotides to the3′ side of the DNA sequence (A) and a nucleotide sequence of 20 to 30nucleotides is ligated to the 5′ side of the DNA sequence (B). Anantisense primer (hereinafter referred to as “primer (F)) complementaryto the primer (E) is synthesized. When a PCR is performed by usingappropriate vector DNA containing DNA (A) as a substrate, sense primer(C) and the chimeric-type antisense primer (F), DNA in which the 20 to30 nucleotides of the 5′ end of DNA (B) is attached to the 3′ end of theDNA (A) can be obtained (the DNA newly formed is designated as DNA (G)).Similarly, when a PCR is performed by using appropriate vector DNAcontaining DNA (B) as a substrate, antisense primer (D) and thechimeric-type sense primer (E), DNA in which 20 to 30 nucleotides of the3′ end of DNA (A) is attached to the 5′ end of the DNA (B) can beobtained (the DNA newly formed is designated as DNA (H)). In the DNAs(G) and (H), the 40 to 60 nucleotides on the 3′ side of the DNA (G) forma sequence complementary to that formed by the 40 to 60 nucleotides onthe 5′ side of the DNA (H). The amplified DNA (G) and (H) are mixed andsubjected to PCR, DNA (G) and (H) are formed into a single strand in afirst denaturation reaction. Although most chains of DNA revert to theiroriginal states following an annealing reaction, a part of DNA formsinto a hetero-double-stranded DNA by the annealing of the complementarynucleotide sequence region. A protruding single stranded part is filledin by a subsequent extension reaction to obtain a chimeric type DNA(hereinafter referred to as DNA (I)) formed of DNA (A) and DNA (B)ligated to each other. DNA (I) can be amplified by performing PCR usingDNA (I) as a substrate, the sense primer (C) and the antisense primer(D). In the present invention, DNA encoding a CDR region of a heavychain and light chain of an anti-human oculospanin mouse monoclonalantibody, DNA encoding an FR region of human immunoglobulin IgG,furthermore DNA encoding a secretion signal of human immunoglobulin IgGmay be used as DNA (A) and (B), on a case-by-case basis, and subjectedto the ligation reaction mentioned above.

Note that codons corresponding to a desired amino acid are known per seand can be arbitrarily chosen. More specifically, the codons can bedetermined in accordance with a customary method in consideration of thefrequency with which the codon is used by a host. A part of nucleotidesequence of the codons may be modified in accordance with a customarymethod such as site-specific mutagenesis (see, Mark, D. F., et al.(1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666) using a syntheticoligonucleotide primer encoding a desired modification. Therefore, ifeach primer is designed so as to introduce a point mutation andthereafter chemically synthesized, it is possible to obtain DNA encodingvariable regions of a heavy chain and light chain of a desiredanti-human oculospanin antibody.

By integrating each of the DNAs of the present invention thus obtainedinto an expression vector, a prokaryotic or eukaryotic host cell can betransformed. Furthermore, by introducing an appropriate promoter and asequence related to phenotypic expression into these vectors, each genecan be expressed in the corresponding host cell.

By virtue of the method mentioned above, a recombinant anti-humanoculospanin antibody can be manufactured easily with high purity and inhigh yield.

6. D A Pharmaceutical Composition Containing an Anti-Human OculospaninAntibody

From the anti-human oculospanin antibodies obtained by a methoddescribed in Section “5. Preparation of anti-human oculospaninantibody”, antibody neutralizing the biological activity of humanoculospanin or an antibody specifically damaging a cancer cellexpressing human oculospanin can be obtained. These antibodies caninhibit the biological activity of human oculospanin in the living body,in other words, canceration of a cell. Therefore, they can be used as amedicament, in particular, as a therapeutic agent for cancer. Theactivity of an anti-human oculospanin antibody in neutralizing abiological activity of human oculospanin in vitro can be determined bythe ability to inhibit canceration of a cell in which human oculospaninis overexpressed. To explain more specifically, the inhibitory activitycan be determined by culturing mouse fibroblast cell strain, NIH3T3,which overexpresses human oculospanin, adding an anti-human oculospaninantibody to the culture system in various concentrations. In this way,the inhibitory activities against focus formation, colony formation andspheroid growth can be determined. The cytotoxic activity of ananti-human oculospanin antibody against a cancer cell in vitro can bedetermined by antibody-dependent cytotoxic activity,complement-dependent cytotoxicity or complement-dependent cell-mediatedcytotoxicity exhibited by the anti-human oculospanin antibody against acell overexpressing human oculospanin. To be more specific, 293T cellsoverexpressing human oculospanin are cultured; then, an anti-humanoculospanin antibody is added at various concentrations to the culturesystem. Mouse spleen cells are further added to the culture system andcultured for an appropriate time. Thereafter, the ratio of induction ofcell death for the cells overexpressing human oculospanin is determined.The effect of an anti-human oculospanin antibody in cancer treatment canbe determined in vivo by using an experimental animal, morespecifically, by administering the anti-human oculospanin antibody to atransgenic animal overexpressing human oculospanin and determining achange in the cancer cells.

An antibody thus obtained for neutralizing the biological activity ofhuman oculospanin or an antibody specifically damaging cancer cellsexpressing human oculospanin is useful as a medicament, especially as apharmaceutical composition for use in cancer treatment or as an antibodyfor use in immunological diagnosis of such a disease. As the type ofcancer, skin cancer and melanoma, a kind of skin cancer, may bementioned; but cancers that can be treated or diagnosed in accordancewith the invention are not limited to these examples.

The present invention provides a pharmaceutical composition containingan anti-human oculospanin antibody in an amount useful for treatment, apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or an auxiliary agent.

A substance to be used as a pharmaceutically acceptable preparation in apharmaceutical composition according to the present invention ispreferably non-toxic to a patient to which the pharmaceuticalcomposition is to be administered, in view of the dose andconcentration.

A pharmaceutical composition according to the present invention cancontain substances, suitable for inclusion in a preparation, which arecapable of changing, maintaining, and stabilizing pH, osmotic pressure,viscosity, transparency, isotonic condition, aseptic condition,stability, solubility, release rate, absorbtion rate, and permeability.Examples of such substances for inclusion in a preparation include, butare not limited to, amino acids such as glycine, alanine, glutamine,asparagine, arginine, and lysine; anti-oxidant agents such asanti-bacterial agents, ascorbic acid, sodium sulfate and sodium hydrogensulfite; buffering agents such as phosphate, citrate, borate buffers,hydrocarbonate, Tris-HCl solution; fillers such as mannitol and glycine;chelating agents such as ethylenediamine tetraacetate (EDTA); complexforming agents such as caffeine, polyvinylpyrrolidine, β-cyclodextrinand hydroxypropyl-β-cyclodextrin; thickening agents such as glucose,mannose, and dextrin; carbohydrates such as monosaccharides,disaccharides, glucose, mannose, dextrin; hydrophilic polymers such ascolorants, flavors, diluents, emulsifiers, polyvinylpyrrolidine;preservatives such as low molecular weight polypeptides, base-formingcounter ions, benzalkonium chloride, benzoate, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid, and hydrogen peroxide; solvents such asglycerin, propylene glycol, and polyethylene glycol; sugar alcohols suchas mannitol and sorbitol; polysorbates such as suspending agents, PEG,sorbitan ester, polysorbate 20, and polysorbate 80; surfactants such asTriton, tromethamine, lecithin, cholesterol; stability-enhancing agentssuch as sucrose, and sorbitol; elasticity-enhancing agents; transportagents, diluents; excipients; and/or pharmaceutical auxiliary agentssuch as sodium chloride, potassium chloride, mannitol/sorbitol. Theamount of these substances added to a preparation is preferably 0.01 to100 times, more preferably 0.1 to 10 times the weight of the anti-humanoculospanin antibody. Those skilled in the art can appropriatelydetermine the formulation suitable for preparation of a pharmaceuticalcomposition depending upon the disease and administration route.

The excipient and carrier used in a pharmaceutical composition may be aliquid or solid substance. Examples of a suitable excipient and carriermay include injectable solutions, saline, artificial cerebral spinalfluid and other substances usually used for parenteral administration.Furthermore, neutral saline and saline containing serum albumin may beused as a carrier. A pharmaceutical composition may contain a Trisbuffer of pH 7.0 to 8.5 and an acetate buffer of pH 4.0 to 5.5, whichmay be supplemented with sorbitol and other compounds. A pharmaceuticalcomposition according to the present invention having a selectedcomposition is prepared with a requisite purity in appropriate drugform, or as a lyophilized product or a liquid product. To describe thismore specifically, a pharmaceutical composition containing theanti-human oculospanin antibody can be formed into a lyophilized productusing an appropriate excipient such as sucrose.

A pharmaceutical composition according to the present invention can beprepared for parenteral use or for oral use for gastrointestinalabsorption. The composition and concentration of a preparation can bechosen depending upon the administration method. As an anti-humanoculospanin antibody contained in a pharmaceutical composition accordingto the present invention exhibits higher affinity for human oculospanin;in other words, the higher the affinity of anti-human oculospaninantibody for human oculospanin, as expressed by the dissociationconstant (Kd value), that is, the lower the Kd value, the higher theefficacy of the pharmaceutical composition of the present invention at alower dose. Therefore, based on this, the dose amount of thepharmaceutical composition of the present invention to a person can bedetermined. The humanized anti-human oculospanin antibody may beadministered to a person as a single dose at an interval of 1 to 30 daysin an amount of about 0.1 to 100 mg/kg.

Examples of forms of a pharmaceutical composition of the presentinvention may include injections such as drip infusions, suppositoryagents, pernasal agents, sublingual agents, and percutaneous absorptionagents.

7. Screening Methods

The present invention provides methods for screening for candidateagents that bind to oculospanin proteins. In some cases, screens aredone for agents that induce cytotoxicity as described herein.

Screening methods can be homogeneous or heterogeneous, with the latterbeing preferred.

Thus, the present invention provides methods of screening candidateagents for agents that bind to and/or modulate the activity of (inparticular, the inducement of cytotoxicity) oculospanin.

“Candidate agent” or “candidate drug” as used herein describes anymolecule, e.g., proteins including biotherapeutics including antibodiesand enzymes, small organic molecules including known drugs and drugcandidates, polysaccharides, fatty acids, vaccines, nucleic acids, etc.that can be screened for activity as outlined herein. Candidate agentsare evaluated in the present invention for discovering potentialtherapeutic agents that affect oculospanin and therefore potentialdisease states.

Candidate agents encompass numerous chemical classes. In one embodiment,the candidate agent is an organic molecule, preferably small organiccompounds having a molecular weight of more than 100 and less than about2,500 daltons. Particularly preferred are small organic compounds havinga molecular weight of more than 100 and less than about 2,000 daltons,more preferably less than about 1500 daltons, more preferably less thanabout 1000 daltons, more preferably less than 500 daltons. Candidateagents comprise functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast one of an amine, carbonyl, hydroxyl or carboxyl group, preferablyat least two of the functional chemical groups. The candidate agentsoften comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression and/orsynthesis of randomized oligonucleotides and peptides. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural or synthetically produced libraries and compounds are readilymodified through conventional chemical, physical and biochemical means.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification to produce structural analogs.

In a preferred embodiment, the candidate bioactive agents are naturallyoccuring proteins or fragments of naturally occuring proteins. By“protein,” as used herein, is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. In some embodiments, the two or more covalently attached aminoacids are attached by a peptide bond. The protein may be made up ofnaturally occurring amino acids and peptide bonds, for example when theprotein is made recombinantly using expression systems and host cells,as outlined below. Alternatively, proteins (for example when used ascandidate agents in screening assays, as outlined below) may includesynthetic amino acids (e.g., homophenylalanine, citrulline, ornithine,and norleucine), or peptidomimetic structures, i.e., “peptide or proteinanalogs”, such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad.Sci. U.S.A. 89:9367, incorporated by reference herein), which can beresistant to proteases or other physiological and/or storage conditions.Such synthetic amino acids may be incorporated in particular whenfragments of oculospanin or antigen binding proteins are synthesized invitro by conventional methods well known in the art. In addition, anycombination of peptidomimetic, synthetic and naturally occurringresidues/structures can be used. “Amino acid” also includes imino acidresidues such as proline and hydroxyproline. The amino acid “R group” or“side chain” may be in either the (L)- or the (S)-configuration. In aspecific embodiment, the amino acids are in the (L)- or(S)-configuration.

Thus, for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eucaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occuring nucleic acids, random and/orsynthetic nucleic acids. By “nucleic acid” or “oligonucleotide” orgrammatical equivalents herein means at least two nucleotides covalentlylinked together. A nucleic acid of the present invention will generallycontain phosphodiester bonds, although in some cases, as outlined below,for example in the use of nucleic acids as candidate agents in screeningassays, nucleic acid analogs are included that may have alternatebackbones, comprising, for example, phosphoramide (Beaucage et al.,Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J.Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579(1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al,Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); and Pauwels et al., Chemica Scripta 26:141 91986)),phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); andU.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem.Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature380:207 (1996), all of which are incorporated by reference). Otheranalog nucleic acids include those with bicyclic structures includinglocked nucleic acids, Koshkin et al., J. Am. Chem. Soc. 120:13252-3(1998); positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. All of these references are hereby expressly incorporated byreference. These modifications of the ribose-phosphate backbone may bedone to facilitate the addition of ETMs, or to increase the stabilityand half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made, as well asmixtures of different nucleic acid analogs, and mixtures of naturallyoccuring nucleic acids and analogs may be made.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides as well as nucleoside and nucleotideanalogs, and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as a nucleoside.

For example, digests of procaryotic or eucaryotic genomes may be used asis outlined above for proteins. In addition, RNAis are included herein.

7. A. Screens

The screens may take on a variety of formats. In general, either thecandidate agents or the oculospanin protein (including fragmentsthereof) are attached to solid supports as described herein. This isgenerally done using any immobilization techniques, including thosedescribed herein, for example through the use of absorbtion to the solidsupport or covalent attachment using functional groups.

In one embodiment, the oculospanin protein is attached to the solidsupport and labeled candidate agents are added, unbound agents arewashed away, and detection of binding of the candidate agent to theoculospanin protein is done. The contacting step is done under reactionconditions that favor agent-target interactions. Generally, this will bephysiological conditions. Incubations may be performed at anytemperature which facilitates optimal activity, typically between 4 and40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high through put screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away.

A variety of other reagents may be included in the assays. These includereagents like salts, neutral proteins, e.g. albumin, detergents, etcwhich may be used to facilitate optimal protein-protein binding and/orreduce non-specific or background interactions. Also reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may beused. The mixture of components may be added in any order that providesfor the requisite binding.

Once a binding event has been detected, the candidate agent may beidentified.

In some embodiments, candidate agents that are identified as binding tothe oculospanin proteins are then added to cytotoxicity assays, asdescribed herein. Alternatively, cytotoxicity assays are run withlibraries of candidate agents without a binding assay done first.

Cytotoxicity assays are generally done as outlined below in theExamples; this generally is done by adding the candidate agent, a cellexpressing oculospanin, and effector cells. The oculospanin-expressingcells can be selected from a number of different cells. In oneembodiment, the oculospanin-expressing cells are naturally occurringcells, such as primary melanoma cells. In some embodiments,oculospanin-expressing cells are cells or cell lines that have beentransformed to produce oculospanin, particularly human oculospanin.

In a preferred embodiment, the methods of the invention utilize arobotic system. Many systems are generally directed to the use of 96 (ormore) well microtiter plates, but as will be appreciated by those in theart, any number of different plates or configurations may be used. Inaddition, any or all of the steps outlined herein may be automated;thus, for example, the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which may be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates;automated lid handlers to remove and replace lids for wells on non-crosscontamination plates; tip assemblies for sample distribution withdisposable tips; washable tip assemblies for sample distribution; 96well loading blocks; cooled reagent racks; microtitler plate pipettepositions (optionally cooled); stacking towers for plates and tips; andcomputer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates,tubes, magnetic particle, or other solid phase matrix with specificityto the assay components are used. The binding surfaces of microplates,tubes or any solid phase matrices include non-polar surfaces, highlypolar surfaces, modified dextran coating to promote covalent binding,antibody coating, affinity media to bind fusion proteins or peptides,surface-fixed proteins such as recombinant protein A or G, nucleotideresins or coatings, and other affinity matrix are useful in thisinvention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,minitubes, deep-well plates, microfuge tubes, cryovials, square wellplates, filters, chips, optic fibers, beads, and other solid-phasematrices or platform with various volumes are accommodated on anupgradable modular platform for additional capacity. This modularplatform includes a variable speed orbital shaker, electroporator, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of the heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 4° C. to 100° C.

In some preferred embodiments, the instrumentation will include adetector, which may be a wide variety of different detectors, dependingon the labels and assay. In a preferred embodiment, useful detectorsinclude a microscope(s) with multiple channels of fluorescence; platereaders to provide fluorescent, ultraviolet and visiblespectrophotometric detection with single and dual wavelength endpointand kinetics capability, fluroescence resonance energy transfer (FRET),SPR systems, luminescence, quenching, two-photon excitation, andintensity redistribution; CCD cameras to capture and transform data andimages into quantifiable formats; and a computer workstation. These willenable the monitoring of the size, growth and phenotypic expression ofspecific markers on cells, tissues, and organisms; target validation;lead optimization; data analysis, mining, organization, and integrationof the high-throughput screens with the public and proprietarydatabases.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells will be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers will facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats may be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In a preferred embodiment, the robotic workstation includes one or moreheating or cooling components. Depending on the reactions and reagents,either cooling or heating may be required, which may be done using anynumber of known heating and cooling systems, including Peltier systems.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit that communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. The general interaction between a central processingunit, a memory, input/output devices, and a bus is known in the art.Thus, a variety of different procedures, depending on the experiments tobe run, are stored in the CPU memory.

7. B. Rational Drug Design

According to another aspect, the present invention is directed to a drugdesign approach for obtaining a substance capable of inhibiting theactivity of human oculospanin based on the tertiary structure of theprotein. This approach is known as a rational drug design method and isused to search for a compound capable of efficiently inhibiting oractivating a function, such as enzymatic activity or binding to aligand, cofactor or DNA. As an example of such a compound, a proteaseinhibitor serving as anti-HIV agent presently marketed is well known. Inanalyzing the three-dimensional structure of human oculospanin accordingto the present invention, a generally well known method such as X-raycrystallography or nuclear magnetic resonance conceivably can be used.Furthermore, in searching for or designing a substance for inhibitingthe function of human oculospanin, a computer-aided drug design method(CADD) can be used. As an example of this case, a low molecular weightcompound (International Publication WO 99/58515) inhibiting the actionof AP-1 is known which is expected to act as a novel genomic drug fortreating chronic rheumatoid arthritis. By virtue of such a method, it ispossible to obtain a substance inhibiting the function of humanoculospanin by directly binding to the human oculospanin or byinhibiting the interaction between the human oculospanin and otherfactors.

Furthermore, according to another aspect, the present invention relatesto a polypeptide associated with human oculospanin of the presentinvention, in other words, a partner protein for controlling theactivity of human oculospanin. More specifically, the present inventionrelates to a screening method for such a partner protein for controllingthe activity of human oculospanin.

One aspect of such a screening method comprises a step of bringing atest protein sample into contact with human oculospanin, therebyselecting a protein binding to the human oculospanin. Such a methodincludes purification of a protein by making use of its affinity forpurified human oculospanin. To describe more specifically, first, asequence formed of 6 histidines is bound to human oculospanin as anaffinity tag. The resultant human oculospanin is incubated in a cellextract solution (that is, a fraction passed through a column chargedwith nickel-agarose) at 4° C. for 12 hours. Then, a nickel-agarosecarrier is separately added to the mixture and the mixture is incubatedat 4° C. for one hour. After the nickel-agarose carrier is sufficientlywashed with a washing buffer, 100 mM imidazole is added to the mixtureto elute a protein specifically binding to human oculospanin andcontained in the cell extract solution. The purified protein is analyzedto determine its structure. A protein that can be purified as describedabove includes a protein which binds directly to human oculospanin and aprotein forming a complex as a subunit with a protein which bindsdirectly to human oculospanin, but having no binding activity for humanoculospanin, thus binding indirectly to human oculospanin [seeExperimental Medicine, Supplementary volume, Biomanual series 5,“Transcriptional factor investigation method” pp 215-219 (published byYodosha Co., Ltd.)].

As alternative methods, there is a cloning method in accordance withFar-Western blot (Experimental Medicine, Supplementary volume, NewGenetic Engineering Handbook, pp76-81, published by Yodosha Co., Ltd.),and a two-hybrid system using a yeast or a mammalian cell (ExperimentalMedicine, Supplementary volume, New Genetic Engineering Handbook,pp66-75, published by Yodosha Co., Ltd.), and “Checkmate mammalian twohybrid system” (manufactured by Promega). However, the present inventionis not limited to use of these methods.

If cDNA of a partner protein directly or indirectly interacting withhuman oculospanin in this manner is available, it can be used infunctional screening of a substance inhibiting the interaction betweenhuman oculospanin and the partner protein. More specifically, a fusionprotein of human oculospanin with glutathione-5-transferase can beprepared. The fusion protein is allowed to bind to a microplate coveredwith anti-glutathione-5-transferase antibody and a biotinylated partnerprotein is brought into contact with the fusion protein. The binding ofthe partner protein with the fusion protein can be detected usingalkaline phosphatase conjugated with streptavidin. When the biotinylatedpartner protein is added, test substances are added at the same time toselect a substance which promotes or inhibits the binding of the fusionprotein and the partner protein. By this method, a substrate directlyacting on the fused protein or a substance directly acting on thepartner protein can be obtained.

When the fused protein binds indirectly to the partner protein viaanother factor, the assay is performed in the presence of a cellextraction solution containing this factor. In this case, a substance,which may act upon the factor, may be selected.

When the partner protein obtained has the activity of suppressing thefunction of human oculospanin, it is possible to screen an anti-canceragent, for example, a useful candidate substance as a therapeutic agentfor prostate cancer, in accordance with a test method using anexpression vector comprising the human oculospanin gene, as describedabove. Furthermore, when the obtained partner protein has the activityof suppressing the function of human oculospanin, a polynucleotidehaving a nucleotide sequence encoding such a suppressor can be used ingene therapy for cancer.

Such a polynucleotide can be obtained by analyzing the amino acidsequence of the identified inhibitor, synthesizing an oligonucleotideprobe comprising a nucleotide sequence encoding the amino acid sequenceand screening a cDNA library or genomic library. Furthermore, in thecase where a peptide having inhibitory activity against a function ofhuman oculospanin is derived from an artificial peptide librarysynthesized at random, DNA comprising a nucleotide sequence encoding theamino acid sequence of the peptide can be chemically synthesized.

In gene therapy, a gene encoding such an inhibitor is integrated, forinstance, into a virus vector and a patient can be infected with a virus(attenuated) comprising the resultant recombinant virus vector. In thebody of the patient, an anti-cancer factor is produced and functions tosuppress proliferation of cancer cells. In this manner, it is possibleto treat cancer.

As a method of introducing a gene therapeutic agent into a cell, both agene transfection using a virus vector and a non-viral gene transfectioncan be used [Nikkei Science, 4, (1994), p. 20-45; Experimental Medicine,Extra number, 12 (15) (1994); Experimental Medicine, Supplementaryvolume, “Basic Technology of Gene Therapy” Yodosha, Co., Ltd. (1996)].

Examples of gene transfection using a virus vector include methods ofintegrating DNA encoding an inhibitor or a mutated version of the DNAinto DNA virus or using a RNA virus such as retrovirus, adenovirus,adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, or sindbis virus and introducing the virus vector into a body. Ofthese, methods using retrovirus, adenovirus, adeno-associated virus, andvaccinia virus are particularly preferred. Examples of non-viral genetransfection include a method of administering an expression plasmiddirectly into the muscle (DNA vaccination method), liposome treatment,lipofection, microinjection, calcium phosphate treatment, and anelectroporation method. Of these, DNA vaccination and liposome treatmentare preferred.

To use a gene therapeutic agent as a medicine in practice, there is anin vivo method for introducing DNA directly into the body, and an exvivo method which comprises taking a certain type of cells out of thebody, introducing DNA into the cells, and returning the cells into thebody [Nikkei Science, 4, (1994), p. 20-45; The Pharmaceutical Monthly,36(1), 23-48 (1994); Experimental Medicine, Extra number 12 (15)(1994)].

When the gene therapeutic agent is administered in accordance with thein vivo method, it is administered through an appropriate administrationroute, such as a vein, artery, subcutaneous tissue, intradermal tissue,or muscle, which differs depending upon the type of disease andsymptoms. When the agent is administered in accordance with an in vivomethod, the gene therapeutic agent is generally prepared in the form ofan injection; however if necessary, a customarily used carrier may beadded. Furthermore, when the agent is prepared in the form of a liposomeor membrane-fused liposome (Sendai virus-liposome, etc.), the liposomeagent may be supplied as a suspension agent, lyophilized agent, orcentrifugally concentrated and lyophilized agent.

A complementary sequence to the nucleotide sequence represented bySequence ID. No. 1 or a complementary sequence to a partial sequence ofthis nucleotide sequence can be used as a so-called antisense therapy.As an antisense molecule, use may be made of DNA partially complementaryto the nucleotide sequence represented by Sequence ID No. 1 of thesequence listing and formed generally of 15 to 30 mer. Also, use may bemade of a stable DNA derivative such as a phosphorothioate derivative,methyphosphonate derivative, or morpholino derivative of the DNA, or astable RNA derivative such as 2′-O-alkyl RNA. Such an antisense moleculecan be introduced into a cell by a method known in the art of thepresent invention, for example by injecting an extremely small amount ofthe antisense molecule, by forming a liposome capsule, or by expressingit by use of a vector having an antisense sequence. Such an antisensetherapy is useful for treating a disease caused by excessive activity ofa protein encoded by the nucleotide sequence represented by Sequence IDNo. 1 of the sequence listing.

A composition containing the antisense oligonucleotide useful as amedicine can be prepared by a known method including mixing apharmaceutically acceptable carrier. Examples of such a carrier and thepreparation method are described in Applied Antisense OligonucleotideTechnology (1998 Wiley-Liss, Inc.). A preparation containing anantisense oligonucleotide can be administered orally by mixing with apharmaceutically acceptable appropriate excipient or diluent, in theform of tablets, capsules, granules, powder or syrup, or administeredparenterally in the form of an injection, suppository, patch, orexternal preparation. These preparations can be prepared by a knownmethod using additives:

excipients including organic excipients such as sugar derivatives (e.g.,lactose, white sugar (sucrose), glucose, mannitol, and sorbitol); starchderivatives (e.g., corn starch, potato starch, α starch, and dextrin);cellulose derivatives (e.g., crystalline cellulose); Arabic gum;dextran; and pullulan; and inorganic excipients such as silicatederivatives (e.g., soft anhydrous silicic acid, synthesized aluminiumsilicate, calcium silicate, and magnesium aluminate metasilicate);phosphates (e.g., calcium hydrogen phosphate); carbonates (e.g., calciumcarbonate), and sulfates (e.g., calcium sulfate);

lubricant agents including metal stearates (e.g., stearic acid, calciumstearate, and magnesium stearate); talc; colloidal silica; waxes (e.g.,beeswax and spermaceti wax), boric acid; adipic acid; sulfates (e.g.,sodium sulfate), glycol; fumaric acid; sodium benzoate; DL leucine;lauryl sulfates (e.g., sodium lauryl sulfate and magnesium laurylsulfate); silicates (e.g., anhydrous silicate, silicate hydrate); andstarch derivatives mentioned above; binding agents includinghydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, and the same compounds as mentioned asexcipients; disintegrating agents including cellulose derivatives (e.g.,low substitution degree hydroxypropylcellulose, carboxymethylcellulose,carboxymethylcellulose calcium, inner-cross-linkedcarboxymethylcellulose sodium; and chemically modified starch celluloses(e.g., carboxymethylstarch, carboxymethylstarch sodium, and cross-linkedpolyvinyl pyrrolidone); emulsifying agents including colloid silica(bentnite and bee gum), metal hydroxides (e.g., magnesium hydroxide andaluminium hydroxide), anionic surfactants (e.g., sodium lauryl sulfateand calcium stearate); cationic surfactants (e.g., benzalkoniumchloride) and non-ionic surfactants (e.g., polyoxyethylene alkylether,polyoxyethylene sorbitan fatty acid ether, and sucrose fatty acidester); stabilizing agents including paraoxy benzoates (e.g., methylparaben, propyl paraben); alcohols (e.g., chloro butanol, benzylalcohol, and phenylethyl alcohol); benzalkonium chloride; phenols (e.g.,phenol and cresol); thimerosal; dehydro acetate; and sorbic acid;flavoring agents including sweeteners, acidic flavors and flavorsgenerally used; and diluents.

As a method of introducing a compound of the present invention into apatient, a colloidal dispersion system may be used in addition to theaforementioned methods. The colloidal dispersion system is expected tocontribute to increasing the stability of the compound in the body andefficiently transporting the compound to a specific organ, tissue orcell. The choice of colloidal dispersion system is not particularlylimited as long as it is generally used, and for example, a lipid-baseddispersion system may be used which includes polymer complexes,nanocapsules, microspheres, beads, or oil-in-water emulsifiers,micelles, micelle mixtures, or liposomes. A preferable colloidaldispersion system consists of multiple liposomes or vesicles of anartificial membrane, which is effective in efficiently transferring acompound to a specific organ, tissue or cell (Mannino et al.,Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem. et Biophys. Acta,1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonnand Cullis, Current Op. Biotech., 1995, 6, 698).

A unilamellar liposome ranging from 0.2 to 0.4 μm in size is capable ofencapsulating a large proportion of macromolecules contained in anaqueous buffer. A compound can be encapsulated in such an aqueous innermembrane and transported to the brain cells in biological active form(Fraley et al., Trends Biochem. Sci., 1981, 6, 77). The liposome isgenerally composed of a mixture of a lipid, particularly a phospholipid,more particularly a phospholipid having a high phase transitiontemperature, with one or more types of steroid, in particular,cholesterol. Examples of a lipid useful for producing a liposome includephosphatidyl compounds such as phosphatidyl glycerol, phosphatidylcholine, phosphatidylserine, sphingolipid, phosphatidylethanolamine,cerebroside, and ganglioside. Of these, particularly useful isdiacylphosphatidyl glycerol in which a lipid moiety has 14 to 18 carbonatoms, in particular, 16 to 18 carbon atoms and is saturated (that is,no double bond is present within the C14-C18 carbon atom chain). Typicalphospholipids include phosphatidyl choline, dipalmitoyl phosphatidylcholine and distearoyl phosphatidyl choline.

The colloidal dispersion system containing liposomes can be used forpassive or active targeting. Passive targeting can be attained using atendency inherent to liposomes, which tend to distribute in thereticuloendothelial system of an organ containing sinusoids.Alternatively, active targeting can be attained by modifying a liposome,for example, by binding a specific ligand thereto, such as viral proteincoat (Morishita et al., Proc. Natl. Acad. Sci. (U.S.A.), 1993, 90,8474), a monoclonal antibody (or its appropriate binding portion),sugar, glycolipid, or protein (or its appropriate oligopeptidefragment); or alternatively, by modifying the composition of theliposome in order to distribute it in organs or cell types other thanthose where liposomes are naturally localized. The surface of thecolloidal dispersion system can be modified in various methods fortargeting. In a delivery system using a liposome as a targeting means,to maintain a ligand for use in targeting by keeping tight associationwith a lipid bilayer, a lipid group is integrated into the lipid bilayerof the liposome. To bind a lipid chain to the targeting ligand, variouslinking groups can be used. Examples of such a targeting ligand bindingto a specific cell surface molecule predominantly found on the cell towhich an oligonucleotide according to the present invention is desiredto be delivered include (1) hormone, growth factor or an appropriateoligopeptide fragment thereof binding to a specific cellular receptorpredominantly expressed by a cell to which delivery is desired; and (2)a polyclonal antibody, monoclonal antibody, or an appropriate fragmentthereof (e.g., Fab; F(ab)′2) specifically binding to an antigenicepitope predominantly found on a target cell. Two or more bio activatorscan be formed into a complex within a single liposome and administered.A medicinal agent for improving intracellular stability and/or targetingability of the contents can be added to the colloidal dispersion system.

Although a therapeutic gene of the present invention can be used in anamount varying with symptom intensity, age, etc. In the case of peroraladministration, the lowermost limit per dose is 1 mg (preferably 30 mg)and the uppermost limit per dose is 2,000 mg (preferably 1,500 mg). Inthe case of injection, the lowermost limit per dose is 0.1 mg(preferably 5 mg) and the uppermost limit per dose is 1,000 mg(preferably 500 mg). Such a dose can be administered subcutaneously,intramuscularly or intravenously.

Now, the present invention will be more specifically described in detailby way of Examples, which should not be construed as limiting thepresent invention. Note that individual operations regarding genemanipulation in the following Examples are performed in accordance withthe methods described in “Molecular Cloning” (by Sambrook, J., Fritsch,E. F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press1989), or performed using commercially available reagents or kits inaccordance with the protocols thereof.

EXAMPLE 1 Screening of a Gene Specifically Expressed in a Cancer Cell

Expression profile analysis was performed, using an EST probe(Affymetrix GeneChip HG-133 probe 223795_at: manufactured by Affymetrix)having a nucleotide sequence partially overlapping with the sequencerepresented by Sequence ID No. 1 of the sequence listing, by use of thedata base (GeneExpress Software System Release 1.4.2) provided byGenelogic company.

Expression of the human oculospanin gene in various cells wasquantitatively compared by considering its transcription. As a result,the expression levels in 8 melanocyte samples were to found to besignificantly high, compared to the levels in other cells samples,including 12 blood-cell samples, 6 glia cell samples, 62 epithelial cellsamples (P values thereof were <0.0001, =0.0007, and <0.0001sequentially in order, FIG. 1, upper panel).

Next, the expression levels of the human oculospanin gene were comparedin samples derived from tissue. More specifically, the amount oftranscription was compared with respect to 66 skin samples from healthyindividuals and 33 melanoma samples. As a result, the amount oftranscription in the melanoma samples was found to be significantly high(P value=0.0001, FIG. 1, lower panel). Furthermore, when skin samplesfrom 66 healthy individuals were compared with 12 melanoma samplesderived from the melanoma skin tissue, the amount of transcription inthe melanoma samples was found to be significantly higher (Pvalue=0.007, FIG. 2, upper panel).

When 66 healthy person's skin samples were compared to 12 melanomasamples derived from lymph node tissue, the amount of transcription inthe melanoma samples was found to be significantly higher (Pvalue=0.0003, FIG. 2, lower panel).

Furthermore, when 13 healthy person's samples derived from lymph nodewere compared to 12 melanoma samples derived from lymph node tissue, theamount of transcription in the melanoma samples was found to besignificantly higher (P value=0.0011, the panel of FIG. 3).

EXAMPLE 2 Acquisition of the Human Oculospanin Gene and Construction ofExpression Plasmid

a) PCR Reaction

As a primer for amplifying human oculospanin cDNA by PCR,oligonucleotides having the following sequences were synthesized inaccordance with a customary method. 5′-CACCATGGAGGAGGGGGAGAGGAGCC(Primer 1, C-3′ Sequence ID No. 5 of the sequence listing)5′-GCCCCGGGCGGGTTTGGCAGCGG-3′ (Primer 2, Sequence ID No. 6 of thesequence listing)

Note that Primer 1 is an oligonucleotide constructed by adding 4 bases,CACC, as a KOZAK sequence, upstream of the initiation codon of the humanoculospanin gene, in other words, an oligonucleotide constructed byadding the 4 bases, the CACC sequence, to the 5′ side of the nucleotidesequence consisting of nucleotides No. 1 to 23 of the Sequence ID No. 1of the sequence listing. The CACC sequence, since it forms a chaincomplementary to the 3′ terminus of the vector when it is integratedinto the cloning vector pENTR/D-TOPO, makes it possible to integrate thegene into the vector whilst maintaining the orientation of the gene.Primer 2 is an oligonucleotide composed of a chain complementary to anucleotide sequence consisting of nucleotides No. 1043 to 1065 of thesequence ID No. 1 of the sequence listing.

The PCR reaction was performed using PLATINUM Pfx DNA polymerase(manufactured by Invitrogen) in accordance with the protocol provided.More specifically, to 0.1 μl of the first strand cDNA obtained, 1.5 μlof each of 10 pmol/μl synthetic primer 1 and synthetic primer 2, 5 μl of10× Pfx Amplification Buffer, 1.5 μl of 10 mM dNTP Mix, 1 μl of 50 mMMgSO₄, 0.5 μg of PLATINUM Pfx DNA polymerase, 10 μl of 10× PCRx EnhancerSolution, and 28.9 μl of sterilized water were added to prepare 50 μl ofa PCR reaction solution. The PCR reaction was performed using a PeltierThermal Cycler TPC-200 DNA Engine (manufactured by MJ Research), firstby heating the PCR solution at 94° C. for 2 minutes, repeating 5 times athermal cycle consisting of reactions at 94° C. for 30 seconds and 65°C. for 2 minutes; 5 times a thermal cycle consisting of reactions at 94°C. for 30 seconds 60° C. for 40 seconds, and 68° C. for one minute and20 seconds; 5 times a thermal cycle consisting of reactions at 94° C.for 30 seconds, 55° C. for 40 seconds, and 68° C. for one minute and 20seconds; 35 times a thermal cycle consisting of reactions at 94° C. for30 seconds, 50° C. for 40 seconds, and 68° C. for one minute and 20seconds and finally maintaining the PCR solution at 68° C. for 10minutes, and then storing the solution at 4° C. A desired cDNA wasobtained by subjecting the reaction product to 1.5% agarose gelelectrophoresis, confirming amplification of the NM_(—)031945 cDNA (1069bp), and purifying the DNA from the agarose gel using the S.N.A.P.UV-Free Gel Purification Kit (manufactured by Invitrogen) in accordancewith the protocol provided. The concentration of cDNA thus purified wasdetermined by use of 1 D Image Analysis Software Version 3.5 (KodakDigital Science EDAS290: manufactured by Kodak) with reference to a 1 kbDNA Ladder which was used as a concentration reference.

b) Cloning of the Human Oculospanin cDNA into the pENTR/D-TOPO Vector

The NM_(—)031945 cDNA obtained in Example 2a) was cloned into thepENTR/D-TOPO vector using the pENTR Directional TOPO Cloning Kit(manufactured by Invitrogen) in accordance with the protocol provided.More specifically, NM_(—)031945 cDNA was mixed with the pENTR/D-TOPOvector, having Topoisomerase bound thereto, in the reaction buffersupplied with the kit and incubated at room temperature for 30 minutes.OneShot TOP10 Chemically Competent E. coli (manufactured by Invitrogen)was transformed using the reaction product obtained and cultured on anLB agar medium containing 50 μg/ml kanamycin. The resultant E. colicolonies, which exhibited resistance to kanamycin, were selected andcultured, in a liquid TB medium containing 1 ml of 50 μg/ml kanamycin,at 37° C. overnight. Plasmid DNA was isolated and purified by using aMontage Plasmid Miniprep₉₆ Kit (manufactured by Millipore). Then, theplasmid DNA thus obtained was subjected to a reaction using the BigDyeTerminator v3.0 Cycle Sequencing Ready Reaction Kit in accordance withthe protocol provided, the nucleotide sequence was analyzed using an ABIPRISM 3100 DNA Analyzer (manufactured by Applied Biosystems). As aresult, it was confirmed that cDNA (Sequence ID No. 1 of the sequencelisting) having an open reading frame of the nucleotide represented byGenBank ACCESSION NO.NM_(—)031945 was integrated into the pENTR/D-TOPOvector.

Next, the gene was transferred to an expression vector, pcDNA3.1/DEST40(manufactured by Invitrogen), using the GATEWAY™ system. To explain morespecifically, 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix (manufactured byInvitrogen), 4 μl of LR Reaction Buffer, 0.3 μg ofpENTR/D-TOPO-NM_(—)031945, and 0.3 μg of pcDNA3.1/DEST40 were mixed andmade up to a 20 μl reaction solution in TE buffer. The reaction solutionwas allowed to react at 25° C. for one hour. After the reaction, 2 μl ofproteinase K was added and a reaction was performed at 37° C. for 10minutes. Using the resulting reaction product, OneShot TOP10 ChemicallyCompetent E. coli (manufactured by Invitrogen) were transformed andcultured in a LB agar medium containing 50 μg/ml of ampicillin. Theresulting E. coli colonies, exhibiting ampicillin resistance, wereselected and cultured in 100 ml of liquid LB medium containing 50 μg/mlampicillin at 37° C. overnight, and plasmid DNA(pcDNA3.1-DEST40-NM_(—)031945) was isolated and purified by use ofPlasmid MAXI Kit (manufactured by QIAGEN).

EXAMPLE 3 Introduction of the Human Oculospanin Gene into Cells,Confirmation that the Human Oculospanin Gene Product is Expressed, andPreparation of a Membrane Fraction from Human Oculospanin ExpressingCells for Use as an Immunogen

a) Transfection of NIH3T3 cells with the plasmidpcDNA3.1-DEST40-NM_(—)031945

NIH3T3 cells were transfected with plasmid pcDNA3.1-DEST40-NM_(—)031945obtained in Example 2 as follows. The transfection of the NIH3T3 cellswas performed by lipofection using the Lipofectamine 2000 Reagentmanufactured by Invitrogen. To explain more specifically, first, NIH3T3cells were grown in a 6 well plate up to a semi-confluent state. Next,the cells were washed once with antibiotic-free DMEM containing 10%fetal calf serum, then 200 μl of antibiotic-free DMEM containing 10%fetal calf serum was added to the cells. Then, to a 1.5 ml Eppendorftube, 100 μl of serum-free medium (DMEM) and 2 μg of plasmid DNA(pcDNA3.1-DEST40-NM_(—)031945) recovered in the aforementioned mannerwere added and mixed. To another 1.5 ml Eppendorf tube, 96 μl ofserum-free medium (DMEM) and 4 μl of Lipofectamine 2000 Reagent wereadded and mixed. The DNA solution and the Lipofectamine solution weremixed and allowed to stand still at room temperature for 20 minutes.Thereafter, the DNA-Lipofectamine solution mixture was added to thecells and cultured at 37° C. in 5% CO₂. After 4 hours, 1 ml of DMEMcontaining 10% fetal calf serum was added to the cells which werecultured at 37° C. overnight in 5% CO₂.

b) Confirmation of Expression of the PlasmidpcDNA3.1-DEST40-NM_(—)031945 in NIH3T3 Cells

The cell culture product thus obtained was recovered. The negativecontrol containing no cDNA or NIH3T3 cells transfected with thepcDNA3.1-DEST40-NM_(—)031945 obtained were washed with a PBS (−) buffersolution (manufactured by Invitrogen). The cells were dispersed in asample buffer solution (manufactured by BioRad) containing2-mercaptoethanol for use in SDS polyacrylamide electrophoresis(SDS-PAGE). SDS-PAGE was performed using 12.5% polyacrylamide gel (ePAGEL E-T12.5L; manufactured by ATTO corporation) under reducingconditions.

After electrophoresis, bands were transferred from the polyacrylamidegel to a Polyvinylidene Difluoride(PVDF) membrane (manufactured byMillipore) by use of a gel-membrane transfer apparatus (NP7513manufactured by Marysol) in a transfer buffer solution (192 mM glycine,20% methanol, 25 mM Tris) under the following conditions: 4° C., 120minutes and 200 mA.

After transfer, the PVDF membrane was subjected to Western blot analysisusing an anti-V5-tag antibody (manufactured by Invitrogen). To explainmore specifically, first, the PVDF membrane was blocked using blockace(manufactured by Yukijirushi Co.,) once at room temperature for 30minutes, and put in a plastic bag (trade name: Hybribag manufactured byCosmo Bio). To the bag, the anti-V5-tag antibody (1000-fold dilution)and 5 ml of blockace were added and the bag was shaken at roomtemperature for one hour. After one hour, the membrane was removed andwashed with PBS containing 0.05% Tween 20 (hereinafter referred to as“0.05% Tween 20-PBS) once at room temperature for 15 minutes and twicefor 5 minutes. Thereafter, the membrane was transferred to a new plasticbag. To the bag, 30 ml of a solution containing a horseradish peroxidaselabeled anti-rabbit IgG antibody (manufactured by Amersham Pharmacia)diluted 5000 fold with 0.05% Tween 20-PBS, was added and shaken at roomtemperature for one hour. After one hour, the membrane was taken out andwashed with 0.05% Tween 20-PBS once for 15 minutes and four times for 5minutes. After washing, the membrane was placed on a wrapping film and aband having the anti-V5-tag antibody bound thereto was detected by useof ECL Western blotting detection solution (manufactured by AmershamPharmacia). The membrane was placed on the wrapping film and soaked inthe ECL Western blotting detection solution for one minute and thenexposed to an X-ray film (one minute). As a result, a band specific tothe NIH3T3 cells having plasmid pcDNA3.1-DEST40-NM_(—)031945 DNAintroduced therein was detected due to the presence of the anti-V5-tagantibody (FIG. 4).

c) Transfection of BALB-3T3 Cells with the PlasmidpcDNA3.1-DEST40-NM_(—)031945

BALB-3T3 cells (American Type Culture Collection No. CCL-163) werecultured in three Cell Trays (culturing area: 500 cm² manufactured bySumitomo Bakelite Co., Ltd.) for cell culture in Dulbecco's ModifiedEagle Medium (hereinafter referred to as “DMEM”) manufactured by NissuiPharmaceutical Co., Ltd., containing 10% bovine serum (hereinafterreferred to as “BS”) manufactured by Gibco), at 37° C. in 5% CO₂ gas upto a semi-confluent state. Thereafter, the BALB-3T3 cells weretransfected with the plasmid pcDNA3.1-DEST40-NM_(—)031945. Thetransfection of the BALB-3T3 cells was performed by lipofection usingGeneporter™ 2 Transfection Reagent (manufactured by Gene TherapySystems). To explain more specifically, the cells were washed once usinga serum-free medium, DMEM. To the cells, 500 ml of the serum-free medium(DMEM) was added. Then, to a 50 ml Falcon tube, 6 ml of New DNA diluentand 240 μg of plasmid DNA (pcDNA3.1-DEST40-NM_(—)031945) recovered bythe aforementioned method were added and mixed. To another 50 ml Falcontube, 4.8 ml of serum-free medium (DMEM) and 1200 μl of Geneporter™ 2Reagent were added and mixed. The DNA solution and the Geneporter™ 2solution were mixed and allowed to stand still at room temperature for20 minutes. Thereafter, the solution mixture with DNA-Geneporter™ 2 wasadded to the cells (4 ml/tray) and cultured at 37° C. in the presence of5% CO₂. After 4 hours, DMEM containing 20% bovine serum was added in anamount of 50 ml/tray and cultured at 37° C. in 5% CO₂ overnight.

d) Preparation of the Cell Membrane Fraction

The cells cultured by the aforementioned method were washed with PBS (−)buffer solution (manufactured by Invitrogen). The cells were collectedusing a cell scraper (manufactured by Sumitomo bakelite Co., Ltd.), andsuspended in 7 ml of 5 mM Tris buffer at pH 8.0. The resulting cellsolution was allowed to stand still at 4° C. for 30 minutes. The cellswere crushed using a Dounce Type B homogenizer (30 strokes) andcentrifuged at 1000 G for 10 minutes. The supernatant was recovered andcentrifuged at 78,000 G for 100 minutes using an ultracentrifugationapparatus (manufactured by Hitachi) and the precipitate was recovered.The precipitate was subjected to a sugar density gradient to concentratethe membrane fragments. More specifically, the precipitate was dissolvedin 3 ml of a solution of 57% sugar and 0.25M Tris buffer, pH 8.0. Theresulting solution was transferred to an ultracentrifuge tube. Analiquot of 3 ml of a solution of 57% sugar and 0.25M Tris buffer, pH 8.0and 1.5 ml of a solution of 37.5% sugar and 0.25M Tris buffer pH 8.0were layered sequentially onto the cell precipitate solution. Then,centrifugation was performed using an ultracentrifugation apparatus at75,500 G for 16 hours. An aliquot of 1 ml was taken from the top of eachtube. To each aliquot (fraction), 10 mL of 5 mM Tris buffer pH 8.0 wasadded and this was subjected to ultracentrifugation at 78,000 G for onehour to recover the precipitate. To the precipitate 500 μl of 5 mM Trisbuffer, pH 8.0 was added and the cell solution was homogenized using aDounce type B homogenizer (10 strokes). The cell membrane fraction wasidentified by Western Blotting method described in the Section“Confirmation of Expression” and used as an immunogen.

EXAMPLE 4 Immunization of Mice and Cell Fusion

(4-1) Immunization

1 ml (total protein amount: 100 μg) of the membrane fraction solution ofthe human oculospanin expressing cells obtained in Example 3 wasinjected intraperitoneally into BALB/c mice which were 4 to 10 weeks old(purchased from Japan SLC Inc.) After two weeks, the same membranefraction solution (20 μg protein/mouse) was injected into the abdominalcavity as a booster immunization.

(4-2) Cell Fusion

The spleen was excised from a mouse at three days after the boosterimmunization and added to 10 ml of a serum-free RPMI 1640 medium (10.4g/l, RPMI 1640 “Nissui” (1): manufactured by Nissui Pharmaceutical Co.,Ltd., hereinafter referred to as “serum-free RPMI medium”) containing 20mM HEPES buffer (pH 7.3), 350 mg/ml sodium hydrogen carbonate, 0.05 mMβ-mercaptoethanol, 50 units/ml penicillin, 50 μg/ml streptomycin, and300 μg/ml L glutamic acid, and the spleen was crushed on the mesh of acell strainer (cell strainer; manufactured by Falcon) using a spatula.The cell suspension solution passed through the mesh was centrifuged tocollect the spleen cells. The spleen cells were washed twice withserum-free RPMI medium, suspended in serum-free RPMI medium and thenumber of cells was counted.

Myeloma cells NSI (American Type Culture Collection TIB-18) werecultured in ASF 104 medium (manufactured by Ajinomoto; hereinafterreferred to as the “serum-containing ASF medium”) containing 10% FCS(manufactured by Gibco BRL) at 37° C. in 5% CO₂ gas such that the celldensity did not exceed 1×10⁸ cells/ml. The myeloma cells thus preparedwere washed with serum-free RPMI medium in the same manner as above andsuspended in serum-free RPMI medium and the number of cells was counted.

The NSI cell suspension solution containing about 3×10⁷ cells and thespleen cell suspension solution containing about 3×10⁸ cells were mixedand subjected to centrifugation, and thereafter the supernatant wascompletely removed. The cell fusion operation below was performed whilstmaintaining the plastic centrifuge tube containing the pellet in abeaker containing hot water at 37° C. To the pellet, 1 ml of 50% (w/v)polyethylene glycol 1500 (manufactured by Boehringer Mannheim) wasslowly added by pipette whilst agitating the pellet using the tip.Thereafter, 1 ml of the serum-free RPMI medium, previously warmed to 37°C., was gently added in twice and a further 7 ml of serum-free RPMImedium was added. After centrifugation, the supernatant was removed and10 ml of hypoxanthine aminopterin thymidine medium (hereinafter referredto as “HAT medium”; manufactured by Boehringer Mannheim) containing 10%FCS was added by pipette whilst gently agitating using the tip. After 20ml of the HAT medium containing 10% FCS was added, the resultingsolution was dispensed to a 96-well cell culture microplate at an amountof 100 μl/well and cultured at 37° C. in 5% CO₂ gas. Seven to eight dayslater, to wells containing medium with a tinge of yellow, fresh HATmedium was added in an amount of 100 μl/well. The fused cells thusobtained were subjected to screening by limiting dilution analysis asmentioned below.

(4-3) Limiting Dilution

The thymus gland was excised from female BALB/c mouse which were 4 to 10weeks old (purchased from Japan SLC Inc.) and crushed on the mesh of acell strainer (Cell Strainer, manufactured by Falcon) using a spatula.The cells passed through the mesh were washed twice with hypoxanthinethymidine medium (hereinafter referred to as the “HT medium”,manufactured by Boehringer Mannheim) containing 10% FCS. The thymusgland cells of the mouse were suspended in 30 ml of the HT mediumcontaining 10% FCS. The suspension solution thus obtained was used as afeeder cell solution. The culture solution containing the fused cellsobtained in Section (4-2) was diluted 10 to 100 fold with the feedercell solution depending upon the cell density and further seriallydiluted with the feeder cell solution until the density of the fusedcells was 5 cells/ml, 1 cell/ml and 0.5 cells/ml. Each of the samplesthus prepared was dispensed into a 96-well cell culture microplate in anamount of 100 μl per well and cultured at 37° C. in 5% CO₂ gas for 5days.

(4-4) Screening

(4-4-1) Cell ELISA

Human oculospanin expressing cells were maintained by culturing them inRPMI 1640 medium (manufactured by Invitrogen) supplemented with 10%fetal calf serum (manufactured by Moregate Biotech), 20 mM HEPES(manufactured by Sigma) and 55 μM 2-mercaptoethanol (manufactured byInvitrogen) at 37° C. in 5% CO₂ gas. Human oculospanin expressing cellsin the logarithmic growth phase were seeded into a cell culture flask ata density of 2×10⁴ cells/cm² and cultured for 3 days. The humanoculospanin expressing cells thus prepared were transferred to a 50 mltube and centrifuged using a HITACHI himac CF8DL at 1,000 rpm for 5minutes (Centrifugation condition 1). The supernatant was removed andthe human oculospanin expressing cells were suspended in a medium.Thereafter, the number of living cells was counted using 0.4% tryphanblue solution (manufactured by Sigma). The density of the live humanoculospanin expressing cells was adjusted using the medium to be 10⁷cells per ml and the resultant medium was dispensed to a 96-wellU-bottom plate in an amount of 100 μl/well. The 96-well U-bottom platewas centrifuged using a HITACHI himac CF8DL at 15,000 rpm for one minute(Centrifugation condition 2). The supernatant was removed using a 200 μltip. The 96-well U-bottom plate was tapped on the side surface tosuspend the human oculospanin expressing cells. To the suspension,hybridoma culture supernatant solutions whose concentrations wereadjusted to 10 μg/ml, 5 μg/ml, 2.5 μg/ml with a medium cooled on ice,were added in an amount of 100 μl/well. Whilst the 96-well U-bottomplate was stirred using a plate mixer (manufactured by Fujirebio Inc.)at intervals of 15 minutes, a reaction was performed at 4° C. for 1.5hours. After completion of the reaction, the 96-well U-bottom plate wascentrifuged under Centrifugation condition 2, and the supernatant wasremoved using a 200 μl tip. A solution (PBS-5% FBS) prepared by adding5% fetal calf serum to PBS(−)(manufactured by Nissui Pharmaceutical Co.,Ltd.) was added to the wells in an amount of 200 μl per well. Afterstirring using a plate mixer, centrifugation was performed underCentrifugation condition 2 and the supernatant was removed using a 200μl tip. Thereafter, the aforementioned operation was repeated twice. The96-well U-bottom plate was tapped on the side surface to suspend thehuman oculospanin expressing cells. To the suspension,peroxidase-labeled anti-human IgG antibody (manufactured by Kirkegaad &Perry Laboratories) diluted 500 fold with PBS-5% FBS cooled in ice wasadded in an amount of 100 μl/well. While the 96-well U-bottom plate wasstirred using a plate mixer at intervals of 15 minutes, a reaction wasperformed at 4° C. for 1.5 hours. After completion of the reaction, the96-well U-bottom plate was centrifuged under Centrifugation condition 2and the supernatant was removed using a 200 μl tip. Then, PBS-5% FBS wasadded in an amount of 200 μl/well and stirred using a plate mixer,centrifuged under Centrifugation condition 2, and then the supernatantwas removed using a 200 μl tip. Thereafter, the aforementioned operationwas repeated twice. The 96-well U-bottom plate was tapped on the sidesurface to suspend the human oculospanin expressing cells. To thesuspension, a color development substrate for peroxidase (manufacturedby Nacalai Tesque Inc.) adjusted to room temperature was added in anamount of 100 μl/well and stirred using a plate mixer for 10 minutes.After centrifugation was performed under Centrifugation condition 2, thesupernatant was transferred to 96-well flat-bottomed plate in an amountof 50 μl/well and absorbance was measured at 405 nm using a plate reader(1420 ARVO multilabel counter, manufactured by PerkinElmer Inc.)

(4-4-2) Flow Cytometry

The human oculospanin expressing cells obtained in Example 3 werecultured and grown in RPMI 1640 medium containing 10% FCS at 37° C. in5% CO₂ gas. A cell suspension solution, prepared so as to contain 1×10⁷cells/ml, was dispensed into 96-well U-bottom microplate (manufacturedby Nunk) in an amount of 50 μl/well and centrifuged (at 90×g, 4° C. for10 minutes). The supernatant was removed and the supernatant of thefused cells cultured in Section (4-3) above was added in an amount of 50μl/well and stirred. The plate was allowed to stand for one hour on ice,subjected to centrifugation (at 90×9, 4° C. for 10 minutes) and thesupernatant was removed. The pellet was washed twice with a flowcytometric buffer solution (PBS containing 5% FCS and 0.04% (w/v) sodiumazide) in an amount of 100 μl/well and 50 μl of 500-fold diluted goatanti-mouse IgG antibody IgG fraction (manufactured by Organon Technica)labeled with fluorescein-5-isothiocyanate (hereinafter referred to as“FITC”) was added as a secondary antibody and allowed to stand still onice for one hour. After centrifugation (at 90×9, 4° C. for 10 minutes),the supernatant was removed. The pellet was washed twice with 100 μl ofthe flow cytometric buffer solution per well, and thereafter 50 μl of a3.7% formalin solution was added and the resulting solution mixture wasallowed to stand for 10 minutes on ice. In this manner, the cells wereimmobilized. After centrifugation (at 90×g, 4° C. for 10 minutes), thesupernatant was removed. The pellet was washed again with 100 μl of theflow cytometric buffer solution per well and suspended in 100 μl of theflow cytometric buffer per well. This was used as a sample for flowcytometry. The intensity of FITC fluorescence emitted from the cells ineach sample was measured using a flow cytometer (Epics Elitemanufactured by Coulter) at an excitation wavelength of 488 nm and adetection wavelength of 530 nm. When the FITC fluorescence intensity ofthe human oculospanin expressing cells exposed to supernatant from thefusion cell culture was much higher (about 100 to 1,000) than that(about 0.3) of the human oculospanin expressing cells unexposed to thesupernatant from the fusion cell culture, the corresponding fusion cellswere selected.

(4-5) Cloning

The cells selected in Section (4-4) above were subjected to a series ofsteps (4-3) to (4-4), five times. In this way, several hybridoma cloneswere obtained which were capable of producing a single antibody capableof binding to human oculospanin expressing cells but incapable ofbinding to the non-transfected parent cells.

EXAMPLE 5 Purification of Human Oculospanin Monoclonal Antibody

Mouse-mouse hybridoma cells constructed in Example 4 were cultured in 1litre of ASF medium containing 10% FCS at 37° C. in 5% CO₂ gas until thecell density reached 1×10⁶ cells/ml. The culture solution wascentrifuged (at 1,000 rpm for 2 minutes), the supernatant was discarded,and the cells collected were washed once using serum-free ASF medium.Thereafter, the cells were resuspended in 1 litre of serum-free ASFmedium and cultured at 37° C. in 5% CO₂ gas for 48 hours. The culturesolution was centrifuged (at 1,000 rpm for 2 minutes) and thesupernatant was recovered and transferred into a dialysis tube(exclusion limit molecular weight: 12,000 to 14,000, manufactured byGibco BRL). Dialysis was performed against a 10-fold amount of 10 mMsodium phosphate buffer solution (pH 8.0). The IgG contained in thesolution within the dialysis tube was crudely purified using highperformance liquid chromatographic apparatus (FPLC system, manufacturedby Pharmacia) under the conditions described below:

Column: DEAE Sepharose CL-6B column (Column size 10 ml, manufactured byPharmacia)

Solvent: 10 mM sodium phosphate buffer solution (pH 8.0)

Flow rate: 1 ml/minute

Elution: 1M sodium chloride linear concentration gradient (0-50%, 180minutes)

The eluate was fractionated into 5 ml samples. The antibody titer of theanti-human oculospanin antibody in each fraction was checked by theELISA method using human oculospanin protein. First, a membrane fractionsolution prepared from human oculospanin expressing cells prepared inExample 3 was added to a 96-well microplate for ELISA in an amount of100 μl/well and kept warm at 37° C. for one hour. Then the membranefraction solution was discarded and each well was washed three timeswith 100 μl of PBS-Tween per well. Then, 100 μl of PBS containing 2%bovine serum albumin was added per well and kept warm at 37° C. for onehour. After washing three times with 100 μl of PBS-Tween per well, 100μl of the elution fraction was added and kept warm at 37° C. for onehour. Furthermore, after wells were washed three times with 100 μl ofPBS-Tween per well, horseradish peroxidase-labeled anti-mouseimmunoglobulin antibody (manufactured by Amersham) diluted 2000 fold inPBS-Tween was added in an amount of 100 μl/well and allowed to react at37° C. for one hour, and then washed three times with 100 μl ofPBS-Tween per well. Subsequently, a substrate for horseradish peroxidase(manufactured by BioRad) was added in an amount of 100 μl/well andallowed to stand still for 5 minutes, and thereafter, the absorbance ofeach well at 415 nm was measured using a microplate reader.

Consequently, the fractions exhibiting high absorbance were collectedand loaded onto two antibody affinity purification columns (HitrapProtein G column, column volume: 5 ml, manufactured by Pharmacia). Afterwashing the inside of the columns with 25 ml of equilibrium buffer (20mM, sodium phosphate buffer (pH 7.0) per column, the antibody was elutedusing 15 ml of an elution buffer (0.1M glycine-hydrochloride (pH 2.7))per column. Each eluate was collected in a test tube containing 1.125 mlof 1M Tris-hydrochloride (pH 9.0). Immediately after completion of theelution, the eluate was loaded onto the upper portion of an ultrafilterof centrifugation-tube form (Centriprep 10 manufactured by Grace Japan)and centrifuged at 3000×g at 4° C. for 2 hours. After the filtratecollected in the lower portion of the filter was removed, 15 ml of PBSwas added to the upper portion and again centrifuged at 3000×g, and 4°C. for 2 hours. In all, this operation was repeated five times. At the5th time of operation, the centrifugation operation was performed untilthe liquid amount in the upper portion of the filter reached 0.5 ml. Theliquid left in the upper portion of the filter was used as a sample ofthe anti-human oculospanin antibody.

EXAMPLE 6 Cytotoxic Activity

Antibody-dependent cytotoxic activity was measured as an index ofbioactivity.

The number of human oculospanin expressing cells (Example 3) was countedby the tryphan blue staining method, the concentration of the cells wasadjusted to 1×10⁶ cells/ml with RPMI 1640 medium (manufactured byInvitrogen, hereinafter referred to as the “RPMI medium”) containing 10%fetal bovine serum (manufactured by Moregate). To the cells, 2.5 μl ofbis(acetoxymethyl)2,2′:6′2″-terpyridine-6,6″-dicarboxylic acid (BATDAlabeling agent, manufactured by PerkinElmer) was added, stirred well andincubated at 37° C. in 5% carbon dioxide for 30 minutes while mixing atintervals of 15 minutes by inverting the culture. To the culture medium,10 ml of the RPMI medium was added, stirred and centrifuged at 1,500 rpmfor 5 minutes. This washing operation was repeated a further two times.The BATDA labeled human oculospanin expressing cells thus obtained wereresuspended in 10 ml of RPMI 1640 medium. An aliquot of 50 μl (5×10³cells) of the suspension solution was seeded in each well of a 96-wellround bottom microplate, which was previously prepared by adding apurified mouse anti-human oculospanin antibody previously adjusted withRPMI 1640 medium to a concentration of 1 μg/ml, or the supernatant ofthe hybridoma culture medium, and leaving it stand still at 4° C. for 30minutes. The microplate was allowed to stand still at 4° C. for afurther 30 minutes. To a negative control well there was added eitherthe purified mouse anti-human oculospanin antibody or RPMI 1640 mediumin place of the hybridoma supernatant.

Effector cells were prepared as follows. J774A.1 cells (available fromDainippon Pharmaceutical Co., Ltd.) were cultured in the presence of 100ng/ml macrophage colony stimulating factor (manufactured by Sigma) for 3days. The number of J774A.1 cells was counted by the tryphan bluestaining method and then adjusted with RPMI medium to a concentration of1×10⁶ cells/ml. To each well of the 96-well round-bottom microplatementioned above, an 100 μl aliquot (1×10⁵ cells) of the cells wasseeded. The microplate was centrifuged at 1,500 rpm for 5 minutes andincubated at 37° C. in 5% CO₂ gas for 4 hours. To a positive controlwell, 1% Triton-X-100 was added in place of the effector cells, in orderto completely kill the BATDA-labeled human oculospanin expressing cells.After a 4 hour incubation, 20 μl of the culture supernatant was takenfrom each well and transferred to 96-well white plate. To the plate, 200μl of a europium solution (manufactured by PerkinElmer) was added. Theplate was shaken at room temperature for 15 minutes and thedecomposition of fluorescence with time was measured.

The rate of cell death induction in each well was calculated based onthe equation below:

Cell death induction rate (%)=(fluorescent count for each testwell−background count for the negative control well)/(the fluorescentcount for the positive control well−background count for the negativecontrol well)×100.

By comparison with a control containing only RPMI 1640 medium, it wasconfirmed that cell death of the human oculospanin expressing cells wasinduced by addition of the purified mouse anti-human oculospaninantibody or the hybridoma supernatant.

EXAMPLE 7 Preparation of Human Oculospanin Expressing Cells and theirMembrane Fraction as Immunogen and Antigen for Detecting Antibody

a) Construction of Plasmid pEF/DEST51-NM_(—)031945

The NM_(—)031945 cDNA obtained in Example 2a) was cloned into thepENTR/D-TOPO vector by using the pENTR Directional TOPO cloning kit(manufactured by Invitrogen) in accordance with the protocol provided.The NM_(—)031945 cDNA was mixed with pENTR/D-TOPO vector havingTopoisomerase bound thereto, in a reaction buffer provided with the kitand incubated at room temperature for 30 minutes. Using the reactionproduct obtained, Oneshot TOP10 chemically competent E. coli.(manufactured by Invitrogen) were transformed and cultured in LB agarmedium containing 50 μg/ml kanamycin. The resulting E. coli colonies,resistant to kanamycin, were selected and cultured in 1 ml of liquid TBmedium containing 50 μg/ml of kanamycin at 37° C. overnight. The plasmidDNA was isolated and purified using a Montage Plasmid Miniprep₉₆ Kit(manufactured by Millipore). Next, the plasmid DNA thus obtained wassubjected to a sequencing reaction performed using a BigDye Terminatorv3.0 Cycle Sequencing Ready Reaction Kit in accordance with the protocolprovided, the nucleotide sequence was analyzed using an ABI PRISM 3100DNA Analyzer (manufactured by Applied Biosystem). As a result, it wasconfirmed that the cDNA (Sequence ID No. 1 of the sequence listing)having an open reading frame of the nucleotide sequence represented byAccession No. NM_(—)031945 was integrated in the pENTR/D-TOPO vector.

Then, the gene was transferred into expression vector pcDNA3.1/DEST40(manufactured by Invitrogen) by use of the GATAWAY™ system. Morespecifically, 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix (manufactured byInvitrogen), 4 μl of LR Reaction Buffer, 0.3 μg ofpENTR/D-TOPO-NM_(—)031945, 0.3 μg of pcDNA3.1/DEST40, were mixed with TEbuffer to prepare a 20 μl solution, which was allowed to react at 25° C.for one hour. After completion of the reaction, 2 μl of Proteinase K wasadded and a reaction was performed at 37° C. for 10 minutes. OneShotTOP10 Chemically Competent E. coli (manufactured by Invitrogen) weretransfected with the reaction product and cultured on LB agar mediumcontaining 50 μg/ml of ampicillin. The resulting E. coli colonies,resistant to ampicillin, were selected and incubated in 100 ml of liquidLB medium containing 50 μg/ml of ampicillin at 37° C. overnight. As aresult, plasmid DNA (pcDNA3.1-DEST40-NM_(—)031945) was isolated andpurified using the Plasmid MAXI Kit (manufactured by Qiagen).

Similarly, the gene was transferred to the expression vector pEF/DEST51(manufactured by Invitrogen) by use of the Gateway™ system. To explainmore specifically, 4 μl of GATEWAY™ LR Clonase™ Enzyme Mix (manufacturedby Invitrogen), 4 μl of LR Reaction Buffer, 0.3 μg ofpENTR/D-TOPO-NM_(—)031945 and 0.3 μg of pEF/DEST51 were mixed with TEbuffer to prepare a 20 μl solution and allowed to react at 25° C. forone hour. After the reaction, 2 μl of proteinase K was added and allowedto react at 37° C. for 10 minutes. OneShot TOP10 Chemically Competent E.coli (manufactured by Invitrogen) were transformed with the reactionproduct obtained and cultured on LB agar medium containing 50 μg/mlampicillin. The resulting E. coli colonies, resistant to ampicillin,were selected and cultured in 100 ml of liquid LB medium, containing 50μg/ml ampicillin, at 37° C. overnight. As a result, plasmid DNA(pEF-DEST51-NM_(—)031945) was isolated and purified using the PlasmidMAXI Kit (manufactured by Qiagen).

b) Transfection of BALB-3T3 Cells and 293T Cells with the PlasmidpEF-DEST51-NM_(—)031945

BALB-3T3 cells (available from RIKEN, clone A31) were cultured in 330150 mm cell-culture dishes (culturing area: 148 cm², manufactured byIWAKI) containing Dulbecco's Modified Eagle's medium (hereinafterreferred to as the “DMEM”, manufactured by SIGMA) supplemented with 10%bovine serum (manufactured by GIBCO; hereinafter referred to as “BS”) at37° C. in 5% CO₂ gas up to a semi-confluent state. Thereafter, theBALB-3T3 cells were transfected with plasmid pEF-DEST51-NM_(—)031945.The Transfection of BALB-3T3 cells was performed by lipofection usingthe Geneporter™ 2 transfection reagent manufactured by Gene TherapySystems. More specifically, the cells were washed once with serum-freemedium (DMEM) and 20 ml of the serum-free medium (DMEM) was added. Then,to a 50 ml Falcon tube, 0.6 ml of New DNA diluent and 24 μg of plasmidDNA (pEF-DEST51-NM_(—)031945) recovered by the aforementioned methodwere added and mixed. To another 50 ml Falcon tube, 0.35 ml of a serumfree medium (Opti-MEM I, manufactured by GIBCO) and 84 μl of Geneporter™2 Reagent were added and mixed. The DNA solution and the Geneporter™ 2solution were mixed and allowed to stand still at room temperature for20 minutes. Thereafter, the solution mixture of DNA-Geneporter™ 2 wasadded to the cells (1 ml/dish) and cultured at 37° C. in 5% CO₂. After 3hours, the medium was replaced with 20 ml of DMEM containing 10% bovineserum per dish and cultured at 37° C. overnight in 5% CO₂.

Furthermore, plasmid pEF-DEST51-NM_(—)031945 was introduced in 293Tcells as follows. Introduction into the 293T cells was performed byusing LIPOFECTAMINE 2000 reagent (manufactured by Invitrogen). The 293Tcells were seeded at a density of 2.5×10⁵ cells/9.2 cm² and cultured at37° C. overnight in 5% CO₂. In a 5 ml polypropylene tube, 10 μl ofLIPOFECTAMINE 2000 reagent and 250 μl of OPTI-MEM I Reduced Serum Medium(manufactured by Invitrogen) were mixed and allowed to react with eachother at room temperature for 5 minutes. In another 5 ml polyethylenetube, 4 μg of plasmid (pEF-DEST51-NM_(—)031945) and 250 μl of OPTI-MEM IReduced Serum Medium were mixed. The LIPOFECTAMINE solution and the DNAsolution were mixed and allowed to react with each other at roomtemperature for 20 minutes. The supernatant was removed from the 293Tcells cultured overnight and an antibiotic-free Dulbecco's ModifiedEagle medium (manufactured by Gibco) containing 10% fetal calf serum(manufactured by Moregate) was added to the cells in an amount of 2ml/9.2 cm². The LIPOFECTAMINE-DNA solution mixture was added to the 293Tcells and incubated at 37° C. in 5% CO₂ gas for 2 days.

c) Preparation of the Cell Membrane Fraction (10 Liter)

The cells cultured by the aforementioned method were washed with a PBS(−) buffer solution (manufactured by Dainippon Pharmaceutical Co., Ltd).The cells were collected using a cell scraper (manufactured by IWAKI)and suspended in 230 ml of a 5 mM Tris buffer solution, pH 7.4. Theresulting cell solution was allowed to stand still at 4° C. for 30minutes. The cells were crushed using a Dounce Type B homogenizer (50strokes) and centrifuged at 1000 G for 10 minutes. The supernatant wasrecovered and centrifuged at 1,000 G for 10 minutes using anultracentrifugation apparatus (manufactured by KUBOTA) and thesupernatant was recovered.

The supernatant was centrifuged at 78,000 G for 100 minutes using anultracentrifugation apparatus (manufactured by BECKMAN) and theprecipitate was recovered. To the precipitate, 14 ml of 57% sucrose inTris buffer was superposed and subjected to sugar density gradient at78,000 G for 16 hours at 4° C. As a result, the membrane fragment of theupper layer was recovered. To the membrane fraction, 55 ml of 5 mM Trisbuffer, pH 7.4, was added and centrifuged at 78,000 G for 60 minutes at4° C. The precipitate was recovered. To the precipitate, 1500 μl of 5 mMTris buffer, pH 7.4, was added and then the cell solution washomogenized by the Dounce type B homogenizer (10 strokes). The membranefraction was identified using a Western blotting method described in theSection “Confirmation of expression”.

EXAMPLE 8 Immunization of Mouse and Cell Fusion

a) Immunization

1×10⁷ cells of the human oculospanin gene expressing cells obtained inExample 7 were injected intraperitoneally into BALB/c female mice whichwere 5 weeks old (purchased from Japan SLC Inc.) After 2, 4, 6 and 8weeks, the human oculospanin gene expressing cells (1×10⁷ cells/mouse)were injected intraperitoneally as a booster in the same manner.

B) Cell Fusion

The spleen was excised out from a mouse on the fourth day after thebooster immunization and added to 10 ml of a serum-free MEM medium (9.4g/L, Eagle MEM medium “Nissui” (1): manufactured by NissuiPharmaceutical Co., Ltd., hereinafter referred to as “serum-free MEMmedium”) containing 10 mM HEPES buffer (pH 7.4), 0.02 mg/l sodiumhydrogen carbonate, and 300 μg/ml L-glutamic acid, and then the spleencells were withdrawn using a 21 G′ syringe and tweezers. The cellsuspension solution was centrifuged to precipitate the spleen cells. Thespleen cells were washed twice with the serum-free MEM medium andsuspended in serum-free MEM medium and the number of cells was counted.

Myeloma cells SP2/0 were cultured in myeloma growth medium (hereinafterreferred to as the “ME medium”) containing 15% FBS (manufactured byGIBCO), 306 μg/ml glutamic acid, and 0.05 mM β-mercaptoethanol at 37° C.in the presence of 7% carbon dioxide gas such that the cell density didnot exceed 1×10⁶ cells/ml. The myeloma cells SP2/0 thus cultured werewashed with the serum-free MEM medium and suspended in serum-free MEMmedium and the number of cells was counted.

The SP2/0 cell suspension solution containing cells, the number of whichcorresponded to about ⅕ of the spleen cells, and the suspension solutionfor the whole spleen cells were mixed. After centrifugation, thesupernatant was completely removed. The cell fusion operation below wasperformed while keeping a plastic centrifuge tube containing the pelletat room temperature. To the pellet, 1 ml of 40% (w/v) polyethyleneglycol 4000 (manufactured by Merck) was slowly added while shaking thecentrifuge tube. Thereafter, 9 ml of serum-free MEM medium previouslywarmed at 37° C. was gently added in three times. After centrifugation,the supernatant was removed and hypoxanthine aminopterin thymidinemedium (hereinafter referred to as the “HAT medium”; manufactured bySIGMA) containing 20% FBS was added using a pipette while gentlystirring with the pipette tip such that the cell density became 2.5×10⁶cells/ml. The HAT medium was dispensed to a 96-well cell-culturemicroplate in an amount of 100 μl/well and cultured at 37° C. in thepresence of 7% carbon dioxide gas. After one day, fresh HAT medium wasadded to all the wells in an amount of 100 μl/well and thereafter, themedium was replaced with fresh medium at intervals of 2 to 3 days. Thefused cells thus obtained were subjected to screening by limitingdilution analysis as mentioned below.

c) Limiting Dilution

The culture solution containing the fused cells obtained in Section (b)above was serially diluted such that the density of fused cells in theHT medium (HY medium in the case of 2nd cloning or later) became 1cell/well (10 cells/ml), and 5 cells/well (50 cells/ml). Each of thesamples thus prepared was dispensed in an amount of 100 μl per well, ina 96-well microplate already containing 100 μl of the HY medium, and themicroplates were cultured at 37° C. in the presence of 7% carbon dioxidegas for 10 days.

d) Screening

d-1) ELISA

The cell membrane fraction obtained in Example 7 was prepared in asolution of 1 μg/ml dispensed into a 96-well EIA plate (manufactured byCOSTAR) in an amount of 50 μl/well. After the plate was allowed to standat 4° C. for one day, the antigen solution within the plate wasdiscarded by shaking well and 80 μl of a solution containing 1% BSA inPBS(−) was added per well. The plate was sealed and stored at 4° C.until use. When used, the plate was returned to room temperature andwashed three times using a Serawasher (manufactured by Bio-Tec) throughwhich PBS (PBS-T) containing 0.1% Tween 20 was supplied. As a primaryantibody, 50 μl of cell culture supernatant obtained after 10 to 12 daysof cell fusion was added to each well and allowed to stand at roomtemperature for one hour. After completion of the reaction with theprimary antibody, the plate was washed three times with PBS-T andalkaline phosphatase labeled anti-mouse IgG antibody (manufactured byBIO SOURCE), diluted 5000 fold with a solution (antigen dilutionsolution) containing 0.5% BSA added to PBS-T, was added to the wells inan amount of 50 μl/well, and allowed to stand still at room temperaturefor one hour. After completion of the reaction with the secondaryantibody, a color-emitting substrate for alkaline phosphatase,p-nitrophenyl phosphate, 2Na6H₂O (pNPP, manufactured by Wako PureChemical Industries Ltd.) returned to room temperature was dissolved toa concentration of 1 mg/ml in pNPP Buffer (97 ml/l diethanolamine, 0.1g/l MgCl₂.6H₂O, pH 9.8) and added to the wells in an amount of 100μl/well. The absorbance was measured at 405 nm and 630 nm using a platereader (manufactured by Nalgene Nunc International)

d-2) Flow Cytometry

HEK293 culture cells obtained in Example 7 were cultured in DMEM mediumcontaining 10% FBS at 37° C. in 5% CO₂ gas. After transfection, thecells were cultured for 24 hours and a cell suspension solution wasprepared so as to contain 2×10⁷ cells/ml. The cell suspension solutionwas dispensed into 96-well V-shape bottom microplates (manufactured byCorning) in an amount of 50 μl/well and subjected to centrifugation(1000×g, 20° C. for 5 minutes). The supernatant was removed and thesupernatant of the fused cells cultured in step (c) above was added atan amount of 50 μl/well, stirred, allowed to stand still on ice for 0.75hours, centrifuged (1000×g, 20° C. for 5 minutes), and then thesupernatant was removed. The pellet was washed twice with a flowcytometry buffer solution (MEM containing 5% FBS) in an amount of 150μl/well. Thereafter, to the pellet, 100 μl of 33-fold diluted rabbitanti-mouse IgG antibody (manufactured by Wako Pure Chemical IndustriesLtd.) labeled with fluorescein-5-isothiocyanate (hereinafter referred toas “FITC”) was added as a secondary antibody, allowed to stand still onice for 0.75 hours, and subjected to centrifugation (1000×g, 20° C. for5 minutes). The supernatant was removed, the pellet was washed twicewith flow cytometry buffer using 150 μl/well and suspended in the flowcytometry buffer in an amount of 500 μl/well. This was used as a samplefor flow cytometry. In each sample, the intensity of FITC fluorescenceemitted from cells was measured by flow cytometry (FC500, manufacturedby BECKMAN) at an excitation wavelength of 488 nm and a detectionwavelength of 530 nm. As a result, the fused cells were selected fromthe sample exhibiting higher FITC fluorescent intensity than those ofHEK293 transient expressing cells to which the supernatant of the fusioncell culture was not added.

e) Cloning

The cells selected in the step (d) above were subjected twice to theoperations of a series of steps c) to d). As a result, several hybridomaclones were obtained which produced a monoclonal antibody which binds toHEK293 transient expressing cells, but does not bind to cells into whichthe anti-human oculospanin expression plasmid has not been introduced.One of the hybridoma strains thus cloned was designated as O3B8-2C9-4F3and deposited at the International Patent Organism Depositary of theNational Institute of Advanced Industrial Science Technology as of Feb.17, 2004 under deposition No. FERM BP-08627.

EXAMPLE 9 Purification of Anti-Human Oculospanin Monoclonal Antibody

The mouse-mouse hybridoma prepared in Example 8 was suspended in HYmedium at a concentration of 1×10⁶ cells/ml and allowed to stand stillat 37° C. in the presence of 7% carbon dioxide for 3 days. The culturesolution thus obtained was centrifuged at 1,600 rpm for 5 minutes. Thesupernatant was recovered and IgG was roughly purified as follows:

Binding buffer: pH 7.0 (20 mM Na₂HPO₄.12H₂O, 20 mM Na₂HPO₄.2H₂O)

Elution buffer: pH 3.0, 100 mM glycine-HCl

Neutralization buffer: pH 9.0, 1M Tris-HCl

A requisite aliquot of Protein G carrier (manufactured by AmershamBiosciences) was taken. After ethanol was removed, the protein G carrieraliquot was washed twice with ultra pure water and washed once with thebinding buffer. The binding buffer was added to the protein G aliquotcarrier to make a 50% gel slurry. The protein G gel slurry was added tothe supernatant of the hybridoma. The resulting mixture was rotated at4° C. for 24 hours and washed three times with the binding buffer. Afterwashing, the elution buffer was added to allow antibody to elute. Theeluate was received by a tube containing neutralization buffer in anamount of 1/10 of the elution buffer. The eluate was loaded onto theupper portion of an ultrafilter of a sample tube (Amicon Ultrafree-MC:manufactured by Millipore) and centrifuged at 5000×g, 4° C. for 20minutes. While the filtrate collected in the lower portion of the filterwas removed, the eluate was added such that the liquid amount in theupper portion of the filter was at least 50 μl. After the whole amountof eluate was added, PBS (−) was added in the volume 3 times as large asthe eluate. In this manner, buffer exchange was performed. The liquidleft in the upper portion of the filter was treated as the anti-humanoculospanin antibody sample.

EXAMPLE 10 Cytotoxic Activity

As an index of biological activity, antibody-dependent cytotoxicactivity was measured. The number of the human oculospanin expressingcells prepared in Example 7 was counted by the tryphan blue stainingmethod and thereafter the concentration of the cells was adjusted withRPMI 1640 medium (manufactured by Invitrogen, hereinafter referred to as“RPMI medium”) containing 10% fetal bovine serum (manufactured byMoregate) to 8×10⁵ cells/0.4 ml. Then 40 μl of Chromium-51 (sodiumchromate manufactured by Amersham Bioscience) was added to the cells,the cells were incubated at 37° C. in 5% CO₂ for 2 hours. To the cells,8 ml of RPMI medium was added, stirred and then centrifuged at 1,500 rpmfor 5 minutes. This washing operation was repeated further twice. TheChromium-51 labeled human oculospanin expressing cells thus obtainedwere resuspended in 4 ml of RPMI medium and seeded in a 96-well roundbottom plate, in which 50 μl of 5 μm/ml purified mouse anti-humanoculospanin antibody adjusted with RPMI medium was already present, inan amount of 50 μl (1×10⁴ cells) per well and allowed to stand still at4° C. for 30 minutes. In a negative control well or backgroundmeasurement well, RPMI medium was added in place of the purified mouseanti-human oculospanin antibody.

Effector cells were prepared as mentioned below. The spleen cells weretaken from BALB/c-nu/nu mouse (female, 7 weeks old) in accordance withthe customary method. Then, the cell number was counted by the tryphanblue staining method, the concentration of the cells was adjusted withRPMI medium to 1.5×10⁷ cells/ml. The cells were seeded into 96-wellround bottom microplates in an amount of 100 μl (1.5×10⁶ cells/ml) perwell, centrifuged at 1,500 rpm for 5 minutes and incubated at 37° C. in5% CO₂ for 4 hours. To the positive control well, 2% Triton-X-100 wasadded in place of the effector cells in order to completely kill theChromium-51 labeled human oculospanin expressing cells. To thebackground measurement well, the RPMI medium was added in place of theeffector cells. Next, incubation was performed for 4 hours, 50 μl of theculture supernatant was taken from each of the wells and transferred toa 96-well Luma Plate (manufactured by PerkinElmer). The plate wasdehydrated at 50° C. overnight, the amount of Chromium-51 in each wellwas measured by a microplate scintillation counter (TopCourt NTX,manufactured by PerkinElmer).

The rate at which cell death was induced in each well was calculated inaccordance with the following formula:

Cell death induction rate (%)=(radioactivity count for each testwell−background count for the negative control well)/(the radioactivitycount for the positive control well−background count for the negativecontrol well)×100.

Compared to the negative control, it was confirmed that addition of thepurified mouse anti-human oculospanin antibody (FIG. 5) induced celldeath in the human oculospanin expressing cells.

INDUSTRIAL APPLICABILITY

By virtue of the present invention, it was found that the expressionlevel of human oculospanin in melanoma is significantly high. Accordingto the present invention, there are provided a method of detectingcancer using the human oculospanin gene and a cancer detection kit, andfurther provided an antibody having cytotoxic activity againstoculospanin and a pharmaceutical composition for treating cancercontaining the antibody.

Sequence list free text

Sequence ID No. 5: PCR sense primer for human oculospanin amplification.

1. An antibody which: a) specifically binds to a human oculospaninprotein selected from at least one member of the group consisting of SEQID NO:2 and SEQ ID NO:4, and b) has cytotoxic activity against a cellexpressing said oculospanin protein.
 2. An antibody according to claim 1wherein said antibody is a chimeric antibody.
 3. An antibody accordingto claim 1 wherein said antibody is a humanized antibody.
 4. An antibodyaccording to claim 1 wherein said antibody is a human antibody.
 5. Anantibody according to claim 1 wherein said antibody is a monoclonalantibody.
 6. An antibody according to claim 1 wherein said antibody isan IgG antibody.
 7. An antibody according to claim 6 wherein said IgGantibody is an IgG1 antibody.
 8. A pharmaceutical composition comprisingan antibody according to claim 1 and a pharmaceutically acceptablecarrier.
 9. A method of screening for binding to human oculospanincomprising: a) contacting a human oculospanin protein with a library ofcandidate agents; and b) determining the presence or absence of bindingof at least one candidate agent and said protein.
 10. A method accordingto claim 9 wherein said protein is immobilized on a solid support.
 11. Amethod according to claim 10 wherein said solid support comprisesmicrospheres.
 12. A method according to claim 9 wherein said candidateagents are labeled.
 13. A method according to claim 12 wherein saidlabel is a fluorophore.
 14. A method according to claim 9 wherein saidcandidate agents are immobilized on a solid support.
 15. A methodaccording to claim 14 wherein said solid support comprises microspheres.16. A method according to claim 9 wherein said oculospanin protein islabeled.
 17. A method according to claim 16 wherein said label is afluorophore.
 18. A method of screening for cytotoxicity induction in apopulation of cells expressing a human oculospanin protein comprising:a) contacting said cells with a candidate agent to form a mixture; andb) assaying for cytotoxicity.
 19. A method according to claim 18 whereinsaid cells are contacted with a library of candidate agents.
 20. Amethod according to claim 18 wherein said candidate agents areantibodies.
 21. A method according to claim 18 wherein said assayingstep includes adding effector cells to said mixture.
 22. A method ofinducing cytotoxicity in a cell expressing human oculospanin comprisingadding an agent that inhibits oculospanin activity such thatcytotoxicity is induced.
 23. A method according to claim 22 wherein saidagent is an antibody.
 24. A method of detecting cancer comprising: a)measuring the amount of nucleic acid encoding oculospanin from a humantest sample; b) measuring the amount of nucleic acid encodingoculospanin from a human healthy sample; and c) comparing the differencein said amounts to determine the presence of cancer in said test sample.25. A method of detecting cancer comprising: a) measuring the amount ofoculospanin protein from a human test sample; b) measuring the amount ofoculospanin protein from a human healthy sample; and c) comparing thedifference in said amounts to determine the presence of cancer in saidtest sample.